High-contrast en bloc staining of whole mouse brain and human brain samples for EM-based connectomics

Abstract

Human cortical gray matter connectomes require high-contrast, uniformly stained specimens with at least 2-3 mm on a side, and mouse whole brain connectomes require specimens with at least 5-10 mm on a side. Here, we report an en bloc staining and post-processing protocol, including dehydration and embedding of neural specimens for high-density neural circuit reconstructions and other applications.

Description

[Technical field]

[0001] FIELD OF THEINVENTION Existing widely used electron microscopy (EM) staining protocols are either limited to small sample volumes, suppress staining gradients for larger structures within the sample, or stain only a subset of these structures, and require charge compensation. To overcome the aforementioned obstacles, we developed a method to uniformly en bloc stain and post-process up to centimeter-sized biological tissue samples (i.e., whole mouse brains). [Background technology]

[0002] background The brain is unique not only in the number of cells it comprises (about 85 billion neurons in the case of the human brain) but also in the extent of direct and specific communication between cells via synaptic connections (each neuron has about 1,000 synaptically connected partner neurons). Mapping the resulting complex connectivity graph is the goal of connectomics.

[0003] A prerequisite for synaptic-resolution connectomics is the dense and uniform deposition of heavy metals in the membranes outlining brain tissue, i.e., neuronal cell bodies, their dendrites, axons, and synaptic vessels, which provides high membrane contrast for electron-based imaging. For decades, the most widely used heavy metal membrane contrast agents included osmium (Os) / uranium compounds, with uranyl acetate (UA) being used to label proteins in the postsynaptic density (PSD) and elsewhere (NPL26, NPL35). The development of "low-osmium" protocols (NPL3, NPL13, NPL37) enabled en bloc staining of tissue samples up to about 100-200 μm thick. However, beyond such sample sizes, large staining gradients resulted, limiting connectomics analysis to smaller samples (NPL2, NPL10).

[0004] To achieve high-contrast staining throughout a large tissue block, the relevant compounds must diffuse from the exterior through numerous successive lipid membranes to the center of the tissue block. Moreover, these compounds must react with target structures within the membrane (or be deposited on the membrane by other means). There is a trade-off between these two goals, since processes that enhance attachment to the membrane reduce the availability of the compound for subsequent diffusion and vice versa. This is especially true when enhancement reactions are employed that further increase compound attachment but reduce diffusivity, such as the application of ferrocyanide to enhance osmium staining. Thus, establishing uniform high-contrast staining in all neurites and synapses of a large tissue specimen is a major challenge for the staining protocols employed, requiring staining procedures on the scale of months rather than days for large specimens (NPL19).

[0005] The development of a modified staining protocol allowed for uniform staining of samples up to approximately 1 mm in size (NPL11), a key step that enables millimeter-sized connectomics data acquisition (Figure 1a). This protocol has since been widely applied (NPL9, NPL16, NPL20, NPL38).

[0006] However, the aspiration to obtain connectomes from even larger samples, especially those involving the deep gray matter of the human cortex (2-3 mm in size), and samples corresponding to brain hemispheres (5 mm in size) and whole brains (10 mm in size) of small mammals such as mice (NPL1) or even humans (NPL21), has revealed the need for improved protocols. Although early attempts at staining whole mouse brains were promising (NPL19), reliable protocols for en bloc staining of 2-3 mm to centimeter-scale samples with high staining contrast do not yet exist. This is particularly challenging as EM imaging and reconstruction have advanced to a stage where large-scale sample analysis is possible (NPL9, NPL12, NPL20).

[0007] Starting from a previously reported 1 mm protocol (NPL11), the inventors have intensively developed a method for uniform, high-contrast en bloc staining of biological tissue samples up to centimeter size and have thus surprisingly found that: - a prolonged initial incubation with a heavy metal compound followed by a reduction reaction enhances the contrast of the membrane (corresponding to step a)); - Reducing the temperature of the reduction reaction protects the sample (corresponding to step c)); - Reducing agents such as ferrocyanide and / or ferricyanide enhance membrane contrast by coordination reactions with osmium species in oxidation state vi; - The tissue contrast is maintained if the 1,2,3-trihydroxybenzene step is performed after washing with HO rather than with a buffered aqueous solution (corresponding to step g)); - replacing thiocarbohydrazide (TCH) with an aqueous solution of 1,2,3-trihydroxybenzene avoids tissue damage (corresponding to step h)); - A stepwise resin infiltration protocol allows for slower polymerization and uniform stability throughout the sample, even in the core. - Omitting the lead-aspartate incubation step results in sufficient conductivity and staining contrast of the sample, overcoming poor diffusivity (after step n).

[0008] The protocol of the present invention may be useful for large-scale connectomics projects in mice and other species. In particular, mouse brain volumes approximate the relevant cortical and subcortical volumes of higher mammals such as non-human primates and humans. The ultimate goal of obtaining a large-scale connectome from parts or the entire human cortex (NPL21) will also benefit from the advances described here, both for basic research and clinical applications. Summary of the Invention [Problem to be solved by the invention]

[0009] The present invention provides a new method for en bloc staining and post-processing of biological tissue samples and its uses. Further preferred embodiments of the invention are defined in the dependent claims. In particular, the present invention solves the technical problem of uniform staining of centimeter-sized neurobiological tissue samples, resulting in high membrane contrast and preservation of the postsynaptic density (PDS) without causing tissue destruction at macro- and micro-level.

[0010] In particular, the method of the present invention provides improved uniformity and contrast when staining biological tissue samples, such as neural tissue, ranging in size from 2-3 mm to 10 mm, without damaging the sample, which was not achieved by previously known methods. Furthermore, the method of the present invention allows access to samples for 3D electron microscopy and subsequent connectomics analysis (e.g., high-resolution connectomics analysis in pathological examination of neural tissue samples). [Means for solving the problem]

[0011] In a first aspect, the present invention relates to a method for staining a biological tissue sample, the method comprising the steps of: a) incubating the sample in a buffered aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% of a heavy metal compound; b) washing the sample in a buffered aqueous solution; c) incubating said sample in a buffered aqueous solution of 1.5-3.5%, preferably 2-3%, more preferably 2.2-2.75%, even more preferably 2.5% reducing agent; d) repeating step b); e) repeating step a); f) repeating step b); g) washing the sample with water; h) incubating said sample in an aqueous solution of 2.5-5.5%, preferably 3-5%, more preferably 3.5-4.5%, even more preferably 4% of a stain amplifier, preferably 1,2,3-trihydroxybenzene; i) repeating step g); j) repeating step a), wherein the heavy metal compound is dissolved in an aqueous solution; k) repeating step g); l) incubating said sample in an aqueous solution of 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4% of a heavy metal compound, said heavy metal compound being different from that in step a); m) further incubating the sample of step l), wherein the temperature is increased to at least 35° C.; and n) repeating step g).

[0012] In a second aspect, the present invention relates to a post-processing method comprising dehydrating and embedding the stained biological tissue sample obtained in step n). In a third embodiment of the invention, a biological sample processed by the presently claimed method is analyzed by electron microscopy.

[0013] In some embodiments of the invention, three-dimensional electron microscopy is preferably by (three-dimensional) high-throughput electron microscopy.

[0014] Some specific aspects of the present invention can be summarized as follows: In some embodiments of the present invention, the heavy metal compound in step a) is osmium tetroxide (OsO4) and the heavy metal compound in step l) is uranyl acetate (UA).

[0015] In some embodiments of the present invention, the reducing agent is ferrocyanide and / or ferricyanide.

[0016] In some embodiments of the present invention, the incubation in step a) is carried out at a temperature of 2 to 30° C. for a period of 3 to 120 hours.

[0017] In some embodiments of the present invention, the washing in step b) is carried out at a temperature below 10° C., preferably at 2-6° C., more preferably at 3-5° C., even more preferably at 4° C., and wherein said washing is carried out for a period of 0.5-190 hours.

[0018] In some embodiments of the invention, the incubation in step c) is carried out at a temperature below 10°C, preferably at 2-6°C, more preferably at 3-5°C, even more preferably at 4°C, and wherein said incubation is carried out for 12-96 hours.

[0019] In some embodiments of the invention, the washing in step d) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said washing is carried out for a period of between 0.5 and 120 hours.

[0020] In some embodiments of the invention, the incubation in step e) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said incubation is carried out for a period of between 1.5 and 72 hours.

[0021] In some embodiments of the invention, the washing in step f) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said washing is carried out for a period of 0.5 to 96 hours.

[0022] In some embodiments of the invention, the washing in step g) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said washing is carried out for a period of between 1 and 96 hours.

[0023] In some embodiments of the invention, the incubation in step h) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said incubation is carried out for a period of between 10 and 64 hours.

[0024] In some embodiments of the invention, the washing in step i) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said washing is carried out for a period of between 1 and 96 hours.

[0025] In some embodiments of the invention, the incubation in step j) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said incubation is carried out for a period of between 5 and 120 hours.

[0026] In some embodiments of the invention, the washing in step k) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said washing is carried out for a period of between 1 and 96 hours.

[0027] In some embodiments of the invention, the incubation in step l) is carried out at low temperature, preferably 2-6°C, more preferably 3-5°C, even more preferably 4°C, and wherein said incubation is carried out for 10-64 hours, and the incubation in step m) is carried out at 40-60°C, preferably 45-55°C, more preferably 50°C, and wherein said incubation is carried out for 1-6 hours.

[0028] In some embodiments of the present invention, the incubation in step m) is carried out at 40-60° C., preferably 45-55° C., more preferably 50° C., and wherein said incubation is carried out for 1-6 hours.

[0029] In some embodiments of the invention, the washing in step n) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said washing is carried out for a period of between 1 and 96 hours.

[0030] In some embodiments of the invention, the samples are dehydrated using a graded ethanol series (step o)).

[0031] In some embodiments of the invention, the stepwise ethanol series ranges from 25 to 100%, preferably varying in 25% steps, with each step occurring for 0.5 to 24 hours.

[0032] In some embodiments of the invention, after step o), the sample is incubated in pure acetone at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C. (step p)).

[0033] More preferably, the sample is embedded by using a low viscosity epoxy resin infiltration method.

[0034] In the low viscosity epoxy resin infiltration method of the present invention, the epoxy resin is preferably gradually increased.

[0035] In some embodiments of the invention, the incubation in step p) is carried out for 2 to 32 hours. The length of the incubation step in step p) can be further adapted to the volume / size of the sample. Usually, the incubation in step p) is carried out for 2 to 32 hours for samples of at least 2 to 3 mm and / or 5 mm (approximately the size of a mouse brain hemisphere) and / or 10 mm (approximately the size of a whole mouse brain) and for 48 to 120 hours, preferably 72 hours, for samples of 10 mm (approximately the size of a whole mouse brain) to be fully embedded (corresponding to the whole-mass embedding protocol).

[0036] In some aspects of the invention, the biological tissue sample is preferably obtained from neural tissue.

[0037] In a preferred embodiment of the present invention, the size of the biological tissue sample is 10 mm, 5 mm, and / or 2-3 mm.

[0038] In another embodiment, the present invention is used for en bloc staining of mouse brain hemispheres, whole mouse brain, cortical, and / or subcortical volumes of higher mammals.

[0039] In some aspects of the present invention, biological samples processed and analyzed by the methods claimed in the present invention are used for (high density) neural circuit reconstruction or connectomics analysis, preferably for high resolution connectomics analysis.

[0040] In some embodiments, the present invention is used for the pathological examination of human neural tissue samples.

[0041] In a further aspect, the present invention relates to a method for staining a biological tissue sample, the method of this aspect comprising the steps of: Aa) incubating the sample in a buffered aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% of a heavy metal compound; Ab) washing the sample in a buffered aqueous solution; Ac) incubating the sample in a buffered aqueous solution of 1.5-3.5%, preferably 2-3%, more preferably 2.2-2.75%, even more preferably 2.5% reducing agent; Ad) washing the sample with the buffered aqueous solution or aqueous salt solution of step Ab), Ae) incubating said sample in an aqueous solution of 2.5-5.5%, preferably 3-5%, more preferably 3.5-4.5%, even more preferably 4% of a stain amplifier, preferably 1,2,3-trihydroxybenzene; Af) repeating step Ad), Ag) repeating step Aa), wherein said heavy metal compound is dissolved in an aqueous or buffered aqueous solution; Ah) washing the sample with an aqueous salt solution; Ai) incubating said sample in an aqueous salt solution of a heavy metal compound at 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4%, said heavy metal compound being different from that in step Aa), Aj) further incubating the sample of step Ai), wherein the temperature is increased to at least 35° C.; and Ak) repeating step Ah).

[0042] In a further aspect, the present invention relates to a method for post-processing the stained biological tissue sample obtained in step Ak), comprising dehydrating and embedding the sample.

[0043] Certain further aspects of the present invention can be summarized as follows:

[0044] In some embodiments of the invention, the heavy metal compound in step Aa) is osmium tetroxide (OsO4) and the heavy metal compound in step Ai) is uranyl acetate (UA).

[0045] In some embodiments of the present invention, the reducing agent in step Ac) is ferrocyanide and / or ferricyanide.

[0046] In some embodiments of the present invention, the incubation in step Aa) is carried out at a temperature between 2 and 30° C. for a period between 3 and 120 hours.

[0047] In some embodiments of the invention, the washing in step Ab) is carried out at a temperature below 10° C., preferably at 2-6° C., more preferably at 3-5° C., even more preferably at 4° C., and wherein said washing is carried out for 0.5-190 hours.

[0048] In some embodiments of the invention, the incubation in step Ac) is carried out at a temperature below 10°C, preferably at 2-6°C, more preferably at 3-5°C, even more preferably at 4°C, and wherein said incubation is carried out for 12-96 hours.

[0049] In some embodiments of the invention, the washing in step Ad) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said washing is carried out for a period of between 0.5 and 120 hours.

[0050] In some embodiments of the invention, the incubation in step Ae) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said incubation is carried out for a period of between 10 and 64 hours.

[0051] In some embodiments of the invention, the incubation in step Ag) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said incubation is carried out for a period of between 5 and 120 hours.

[0052] In some embodiments of the invention, the aqueous salt solution is KCl or NaCl.

[0053] In some embodiments of the invention, the washing in step Ah) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said washing is carried out for a period of between 1 and 96 hours.

[0054] In some embodiments of the invention, the incubation of step Ai) is carried out at low temperature, preferably at 2-6°C, more preferably at 3-5°C, even more preferably at 4°C, and wherein said incubation is carried out for 10-64 hours.

[0055] In some embodiments of the present invention, the incubation in step Aj) is carried out at 40-60° C., preferably 45-55° C., more preferably 50° C., and wherein said incubation is carried out for 1-6 hours.

[0056] In some embodiments of the invention, the washing of step Ak) is carried out at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C., and wherein said washing is carried out for a period of between 1 and 96 hours.

[0057] In some embodiments of the invention, after step Ak), the sample is dehydrated, preferably by incubating the sample in a graded ethanol series (step A1).

[0058] In some embodiments of the invention, the stepwise ethanol series of step A1) ranges from 25 to 100%, preferably varying in steps of 25%, and wherein each step is carried out for 8 to 24 hours.

[0059] In some embodiments of the invention, after step A1), the sample is incubated in pure acetone at a temperature higher than 4° C., preferably between 4 and 30° C., more preferably between 15 and 25° C., even more preferably at 25° C. (step Am)).

[0060] In some embodiments of the invention, the incubation in step Am) is carried out for 32 to 48 hours.

[0061] In some embodiments of the present invention, after step Am), the sample is embedded by using a low viscosity epoxy resin infiltration method.

[0062] In some embodiments of the present invention, the concentration of the epoxy resin in step Am) is gradually increased.

[0063] In some embodiments of the invention, the biological tissue sample is derived from neural tissue, preferably selected from the group consisting of higher mammalian cerebellum and human cortical tissue samples.

[0064] In some embodiments of the present invention, the human cortical tissue sample has a size of 2-4 mm in plane (base region) and 2-5 mm in thickness, preferably 2-3 mm in plane (base region) and ≦2 mm in thickness.

[0065] In some embodiments of the invention, the biological tissue sample is analyzed by electron microscopy, preferably three-dimensional electron microscopy.

[0066] In some embodiments of the invention, the three-dimensional electron microscopy is preferably high-throughput electron microscopy.

[0067] In some aspects of the present invention, said biological samples processed and analysed by the methods claimed in the present invention are used for (high density) neural circuit reconstruction or connectomics analysis, preferably for high resolution connectomics analysis.

[0068] In some embodiments, the present invention uses human neural tissue samples for pathological examination. [Brief description of the drawings]

[0069] DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS In the following, preferred embodiments of the present invention will be described in detail with reference to the drawings. [Figure 1-1]High-contrast staining protocols for 2-3 mm sized specimens. (a), (b) Staining gradients have been a major constraint on specimen size for decades. Early reduced osmium thiocarbohydrazide (TCH) protocols (NPL3, NPL13) gave high-contrast uniform staining for specimens up to about 200-250 μm thick. Heavy metal nuclei were provided primarily by osmium and uranium compounds, enhanced by TCH (NPL29) and ferrocyanide (NPL3, NPL13, NPL37). A modified protocol from Hua et al., 2015 (NPL11) allowed staining of specimens up to 1 mm in size. The protocol reported here extends this to 2-3 mm sized specimens, large enough to cover the depth of the human cortex. (c) Staining a 2 mm sized specimen using the Hua et al., 2015-protocol (NPL11) results in a staining gradient over 1 mm, with a low contrast core. From left to right: overview image showing the three-layer gradient; high-resolution EM image of the outer position of the specimen. Note that the contrast is comparable to a 1 mm specimen; the contrast drops sharply in the middle; the signal is too small in the center to obtain a meaningful image. (d), (e) In this protocol, these gradients were eliminated. See Table 1 for the protocol steps and their relevance; note that extending the initial OsO4 incubation time further improves the contrast (e). (f), (g) Temperature-dependent resin infiltration rate. Resin infiltration of larger specimens requires the separation of diffusion and polymerization to avoid a soft core in specimen (f). Using an appropriate infiltration protocol (Table 2) can avoid a soft core. (h) Quantification of membrane contrast depending on location within the sample for the Hua protocol (NPL11) (c), the current 2 mm protocol-Os3 (d) and Os24 (e), quantified as pixel intensity difference between membrane and non-membrane voxels (see Example 1, section "Quantification of membrane contrast").It should be noted that contrast differed significantly between conditions (2-way ANOVA and Tukey's multiple comparisons, see Methods), being higher in the mid and core locations with this protocol (post-hoc one-tailed t-test, n=4.75×106 voxels, p<10-4), and not decreasing depending on location within the specimen with this protocol (Tukey's multiple comparisons, p>0.4). [Figure 1-2] Same as above [Figure 1-3] Same as above [Figure 1-4] Same as above [Figure 2-1] High-contrast staining protocol for mouse brain hemispheres. (a) Size of mouse brain hemisphere compared to the 2–3 mm block size reported in Figure 1, and micro-computed tomography (μCT) images of mouse brain hemispheres used for protocol development. (b)-(d) The main challenges for staining of brain hemispheres and whole mouse brains were sufficient OsO4 penetration without overexposure of OsO4 in the periphery, including the highly relevant gray matter of the cerebral cortex (b); and damage to the specimen on the macro (c) or micro (d) scale. Only macro-damage could be clearly identified in μCT, but also micro-damage would be disruptive for the purpose of whole brain connectomics analysis. (e) To assess the quality of staining, stained brain hemispheres were cut coronally into four blocks, which were then embedded separately. The block containing the center of the brain hemisphere was cut in half, and the quality of staining was examined by SEM. (f) Mouse brain hemisphere (H5) stained uniformly using the protocol developed here (see Tables 1 and 4, experiment 19: steps b-c performed at room temperature), note the high contrast from cortical to subcortical locations (approximate location of high-resolution EM image shown in μCT overview). (f') Mouse brain hemisphere (H13) stained uniformly using the protocol developed here (see Tables 1 and 4, experiment 20: steps b-c performed at 4 °C), showing uniform high-contrast staining of cortical and subcortical locations and preservation of white matter myelin axons (approximate location of high-resolution EM image shown in μCT overview). To show the quality of the staining, the high-resolution image had a pixel size of 5.62 × 5.62 nm2 in f and an electron dose of 113 e- / nm2. [Figure 2-2] Same as above [Figure 2-3] Same as above [Figure 2-4] Same as above [Figure 2-5] Same as above [Figure 3-1] High-contrast staining protocol of whole mouse brain. (a) Size of whole mouse brain and μCT section (sagittal plane, bottom; transverse plane, right) of whole mouse brain completely and uniformly stained using the protocol developed here (Table 1). (b) Whole brain after staining was first cut coronally into four blocks, which were then embedded separately. The block containing the center of the whole brain was cut in half, and the quality of staining was examined by SEM. (c) High-resolution EM images acquired at various locations from the superficial cortex to the subcortical tissue of the mouse brain, the approximate locations are indicated in the μCT overview (top left). To show the quality of staining, the high-resolution images had a pixel size of 5.62 × 5.62 nm2 and an electron dose of 113 e- / nm2. [Figure 3-2] Same as above [Figure 4-1]Analysis of the staining gradient of a 2-3 mm specimen. (a) Left: μCT image and scanning electron microscopy (SEM) confirmation (right) of the staining gradient generated in a 2 mm specimen by the Hua protocol (NPL11). (b) EDS analysis of the staining gradient of the specimen in (a). (c) μCT diagnosis of the staining gradient after each successive step of the Hua staining protocol (NPL11) showing that a gradient occurred after the FeCN and TCH steps. (d) Extending the FeCN step from 3 to 12 h eliminated the FeCN-related gradient. (e) Extending the TCH incubation severely damaged the specimen. (f) Omission of the TCH step resulted in very low specimen conductivity; meaningful images could not be obtained in a high-vacuum SEM due to specimen charging. (g) Replacement of TCH with pyrogallol (Pg) (NPL19) restored specimen conductivity. (h) EDS analysis shows that PG incubation resulted in the final osmium concentration in the sample reaching the same level as with TCH. (i) Comparison of pyrogallol-related steps in water (top) and CaC (bottom). Using CaC buffer reduced the final contrast and also observed a gradient. Electron dose: 18e- / nm2 for high-resolution EM images of (f), (g), and (i). (j) Effect of duration of pyrogallol-related steps. Left: Pyrogallol-related gradient depends on the location of the tissue sample, being stronger in myelin-rich subcortical regions; right: Prolonged pyrogallol incubation and a third OsO4 step eliminated the gradient at all sample positions. (k) Gradients associated with UA and Ld incubation. UA-related gradient can be eliminated by increasing the concentration from 2% to 4%. Ld gradient cannot be eliminated due to limited solubility and is omitted. [Figure 4-2] Same as above [Figure 4-3] Same as above [Figure 4-4] Same as above [Figure 4-5] Same as above [Figure 5-1]Effect of time and temperature of Os and FeCN incubation on membrane contrast in 2–3 mm samples. (a) Membrane contrast was not enhanced in samples stained with 3 h OsO4 (left) or 3 h OsO4 followed by 17 h FeCN. (b) Membrane contrast was not enhanced in samples stained with 24 h OsO4 (left), but was enhanced in samples stained with 24 h OsO4 followed by 17 h FeCN. (c) Incubating OsO4 at room temperature for 3 (c) and 6 (d) days, respectively, improved membrane contrast but also significantly increased background extraction. (e) Incubating OsO4 at 4 °C for 7 days avoided background extraction but reduced contrast. (f) Incubating OsO4 at 4 °C for 6 days followed by 1 day at room temperature avoided background extraction and gave good contrast. (g) Incubation of OsO4 at 4°C for 6 days followed by incubation at room temperature for 1 day and then incubation of FeCN at room temperature for 1 day destroyed the ultrastructure. (h) Incubation of OsO4 at 4°C for 6 days followed by incubation at room temperature for 1 day and then incubation of FeCN at 4°C for 1 day restored the ultrastructural quality. (i) Enhanced membrane contrast was also observed in a sample stained with OsO4 at 4°C for 7 days followed by staining with FeCN at room temperature for 1 day. (j) Enhanced membrane contrast was also observed in a sample stained with OsO4 at 4°C for 7 days followed by staining with FeCN at 4°C for 1 day. All images were taken in a low vacuum SEM at 30 Pa with a maximum electron dose of 70 e- / nm2. [Figure 5-2] Same as above [Figure 5-3] Same as above [Figure 5-4] Same as above [Figure 6-1]Gradients and breakage in mouse brain hemispheres. (a) Observation of OsO4 diffusion into mouse brain hemisphere samples at consecutive time points at 4 °C using μCT. (b) OsO4-FeCN incubation resulted in a macroscopic double gradient with reduced staining intensity in the outer and innermost parts of the sample. (c) A buffer rinse step between OsO4 and FeCN avoided the OsO4-FeCN double gradient. (d) Other than creating a double gradient, OsO4-FeCN incubation slows down FeCN diffusion and does not cause membrane contrast enhancement in the center of the sample. (e) A buffer rinse step between OsO4 and FeCN speeds up FeCN diffusion and enhances membrane contrast in the center of the sample. (f) EDS measurements of the center of the samples in (d) and (e) reveal the penetration depth of FeCN in (c) and (d). (g) When the buffering step is 1 or 2 days at 4 °C, OsO4 is not easily washed away and a gradient (top) is created that is amplified by Pg and also causes damage (bottom) (note also the CaC interaction in H1 because the H2O rinse before Pg was not long enough). (h) When the buffering step is 2 days at room temperature or 4 days at 4 °C, OsO4 is washed away and a gradient is created both before Pg (top) and after Pg (bottom). (i) Adding an OsO4 incubation step after FeCN improves the stability of the sample in H2O up to 100 hours. All SEM images were taken with a low-vacuum SEM at 30 Pa and a maximum electron dose of 70 e- / nm2. [Figure 6-2] Same as above [Figure 6-3] Same as above [Figure 7] Reproduction of mouse brain hemisphere staining. (a), (b) Brain hemispheres H4 and H6 stained as in Table 1, Table 4. (c) Brain hemisphere H13 stained as in Table 1, Table 4. A total of n=4 brain hemispheres, together with H5 (Figure 2) and H13, were successfully stained using the parameters reported in Table 1, Table 4. Also note that H13 shows better preservation of postsynaptic density (PSD). All SEM images were taken in high vacuum with a maximum dose of 84e- / nm2. [Figure 8-1]Experiments carried out to investigate possible chemical mechanisms underlying staining contrast generation. (a), (b) When an intermediate CaC washing step was added between OsO4 and FeCN, the final contrast remained similar to the 2 mm protocol (Fig. 1(c), (d)). Imaged by SEM at high vacuum, electron dose 18e- / nm2. (c) Staining with potassium osmate(vi) produced precipitation bands but no contrast enhancement. (d) UV-vis spectra of potassium osmate(vi) in sodium cacodylate buffer over 24 h. (e), (f) 2 mm samples stained for 3 h and 24 h with the "reduced osmium" protocol. (g) Addition of CaCl2 to OsO4 abolished the membrane contrast enhancement of FeCN. (h) OsO4 incubation at 4 °C also abolished the membrane contrast enhancement effect of FeCN. SEM acquisition of panels (c), (e), (f), (g), and (h) was performed at a low vacuum of 30 Pa and a maximum electron dose of 70 e- / nm2. [Figure 8-2] Same as above [Figure 8-3] Same as above [Figure 9-1]Possible chemical logic behind the staining results. (a) Comparison of bright-field color appearance of samples stained with OsO4 for 2 h, 24 h, 24 h (dilution to assess color), and OsO4 (24 h) → CaC → FeCN (intermediate CaC wash to remove OsO4), top row. For comparison, the pure staining solutions are shown in the bottom row: Os(vi), and the in vitro reaction of Os(vi) with FeCN (bottom). Note that the sample solution after incubation in OsO4 for 24 h looks similar in color to the Os(vi) solution (pink); and the sample solution after Os-CaC-FeCN staining looks similar in color to the in vitro Os(vi) + FeCN reaction (blue-green). (b) Left: Raman spectrum of the product of Os(vi)+FeCN showing a shifted Os(vi) peak, indicating a potential coordination reaction between Os(vi) and FeCN; center: However, in the wavelength range corresponding to FeCN(ii) and FeCN(iii), we found only signals consistent with the FeCN(ii) peak, but not FeCN(iii), suggesting that the reaction between Os(vi) and FeCN was not a redox reaction. (c) The same Os(vi) signal was observed in the brain staining solution after FeCN staining and the in vitro Os(vi)+FeCN reaction. (d) Schematic of the possible chemical reaction. A short incubation of OsO4 ("Os short") could result in Os deposition both on the membrane and in the background (mainly on proteins in the cytoplasm). Os(vi) would be generated on the membrane surface and within the extracellular matrix. If FeCN is then applied to the sample, it will donate CN- as a ligand to Os(vi); however, this will only happen to the Os(vi) at the membrane surface, since the background Os(vi) is not yet accessible to FeCN. This results in osmium being retained both in the membrane and in the background, and no contrast enhancement occurs. However, if the initial OsO4 incubation is prolonged ("Os long"), the over-oxidation of OsO4 will cause a change in the 3D conformation of the background proteins, exposing the hidden Os(vi) to the aqueous phase. If FeCN is then applied, it will donate CN- to Os(vi) both at the membrane surface and in the cytoplasm and extracellular matrix.As a result, osmium remains only within the membrane, thus increasing the local contrast. (e) Summary of Os(vi) coordination chemistry potentially relevant to the staining mechanism. Performed in e- / nm2. [Figure 9-2] Same as above [Figure 9-3] Same as above [Figure 10-1] Alternative protocol approaches for highly sensitive samples. (a) Overview of experiments performed to investigate alternatives to incubation in normal HO near the Pg step that may result in hyperosmotic stress. Briefly, brain hemispheres were first stained with our final brain hemisphere protocol, but only from the first Os to the CaC rinse after FeCN. Alternatives to control for the presence of Os(vi) before the Pg step were then explored (see Example 3, section "Additional variant protocols for highly fragile samples"): (b, d) versus (c, e). (b) Using CaC between the second Os and Pg (Os(vi) present) resulted in a staining gradient appearing at the Pg step. (c) Omission of the second Os step (Os(vi) absent) also resulted in the disappearance of the staining gradient at the Pg step. (d) Similar to c, stabilizing Os(vi) using KCl between the second Os and Pg resulted in a staining gradient appearing during Pg. (e) If Os(vi) was removed by temporarily adjusting the pH of KCl to 1 (instead of canceling the second Os), no staining gradient would occur at the Pg step. (f) Further staining of the sample from c with Os in water gave acceptable membrane contrast. (g) Further staining of the sample from c with Os in CaC gave acceptable membrane contrast. (f, g) Imaged with low vacuum SEM (60pa, beam current ~55pA), pixel size 11.24nm, electron dose ~25e- / nm2. These experiments show alternative protocols for very fragile samples, either omitting the second Os step or using other tested means to avoid pure H2O incubation. e- / nm2 were performed. [Figure 10-2] Same as above [Figure 10-3] Same as above [Figure 11]Staining of a human brain biopsy sample several mm thick spanning the cortical thickness. (a) Immersion fixation, vibratome sectioning, and specimen preparation from a human brain biopsy from a neurosurgical specimen (same operations as in (NPL40)). (b) Human brain sample processed using this protocol for large volume staining (here, 2 x 3 x 2 mm block = 12 mm3). (c) EM overview of a 3 mm tall tissue block covering the pia mater and L1-6 of a human cortical sheet (left) and high resolution EM images of different locations along the cortical axis showing high quality staining. Note that an ATUM ultrathin section series was also successfully cut from this sample, showing the penetration quality. Pixel size in c: 11.24 nm; electron dose 42 e- / nm2. Electron dose 42 e- / nm2 was performed. [Figure 11-2] Same as above [Figure 11-3] Same as above [Figure 12-1] 3D EM of a fully embedded mouse brain hemisphere and quantification of neurite reconstructions. (a) Staining and whole-volume embedding of a mouse brain hemisphere; cut in half in the middle to obtain 3D EM stacks. (b) SEM imaging of different locations on the block face showing uniform high-contrast staining in the cortical and subcortical regions. (c) Trimming the brain hemisphere block into nine parts and using the central block to obtain an SBEM stack (located in the striatum). (d) SBEM dataset (pixel size 11.24 × 11.24 nm2, cut thickness 35 nm). Twenty randomly seeded axons (from a bounding box of size 3 × 3 × 3 μm3) were manually reconstructed in the entire dataset. For comparison, an SBEM dataset of mouse striatum from a conventional 1 × 1 × 0.5 mm3 sized specimen stained using the Hua protocol (NPL11) was analyzed in the same way. The mean inter-error distance of axon reconstructions was similar between the brain hemisphere stained striatal dataset and the control striatal dataset. (e) Automated high-density axonal reconstruction (method described in (NPL40)) yielded similar axonal path length distributions between the striatal hemisphere and the control striatal dataset. Pixel size (b,c) 5.62nm, (d) 11.24nm. Electron dose: (b,c): 113e- / nm2. 3D stack acquisition electron dose: 28e- / nm2. e- / nm2 was performed. [Figure 12-2]Same as above [Figure 13] Summary of artifacts remaining in whole brain staining protocols. (a) μCT cross-section of H5 showing the location of the artifacts shown in detail in b–e. (b) Red arrow: “vascular artifact”: disruption of vascular pericytes from the surrounding neuropil. (c) Vascular artifacts visible in μCT images of 2 mm specimens stained with OsO4 for 1 day at room temperature or 4 days at 4 °C. (d, e) Yellow arrows: micro-breaks less than 1 μm wide were occasionally observed in subcortical regions, especially in densely myelinated areas. (f) During H2O incubation, the cerebellum was particularly sensitive to macroscopic damage. (g) Changing the CaC rinse step between the first OsO4 and FeCN to 4 °C improved the stability of the cerebellum in whole brain staining. All SEM images were acquired in high vacuum with a maximum dose of 21 e- / nm2. [Figure 14-1] 3D EM from a whole embedded mouse brain. (a) A whole brain (W3) was first stained and fully embedded using the protocols described herein (Tables 1 and 2 for staining and embedding procedures, respectively). The whole brain after whole embedding was cut in half and the quality of the staining was examined by SEM. (b) Ultrathin sections of 38 nm were cut from the center of a fully stained and fully embedded whole mouse brain and high-resolution EM images were acquired at various locations along the mouse brain from the superficial cortex to the subcortical tissue, the approximate locations are indicated in the μCT overview (top left). To show the quality of the staining, high-resolution images were acquired with a pixel size of 5.62 × 5.62 nm2 and an electron dose of 113 e- / nm2. (c) After cutting in half, the exposed surface of the whole brain specimen was trimmed into a hexagon (approximately 4 × 2 mm) to test ATUM cutting. Two trials were performed. In the second ATUM trial, the top 1 mm of the surface was trimmed and a new hexagon was created. (d) Photograph of the ATUM cutting run. (e) Photograph series of the ATUM cutting run showing a complete cutting cycle, during which ultrathin sections with a thickness of 38 nm were produced. e- / nm2. [Figure 14-2] Same as above [Figure 14-3] Same as above DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0070] Detailed Description of the Invention The present invention provides a method for en bloc staining and post-processing of biological tissue samples for electron microscopy. In particular, the method of the present invention relates to uniform and high-contrast staining of neural tissues up to the centimeter range without damaging the sample at the macro- and micro-level, which was not achievable with previous methods. With the method of the present invention, neural tissues including cortical volumes with a size of 2-3 mm, mouse brain hemispheres with a size of 5 mm, and whole mouse brains with a size of 10 mm were stained uniformly without detectable staining gradients or breakage (see Example 2). Furthermore, the method of the present invention relates to uniform resin infiltration during embedding of the sample, which improves sample stability even in the sample center. The method of the present invention allows obtaining high quality samples for electron microscopy and subsequent high-resolution connectomics.

[0071] In a first aspect, the present invention relates to a method for staining biological tissue samples. The present invention relates to a chemically rational staining protocol that includes heavy metal deposition / uptake by incubation with heavy metals and a reaction to enhance it. Optionally, the sample is washed extensively with (buffered) aqueous solutions and water to wash away previous reagents that may interfere with the reagents of the following steps.

[0072] The method of the first aspect of the invention comprises the steps of: a) incubating the sample in a buffered aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% of a heavy metal compound; b) washing the sample in a buffered aqueous solution; c) incubating the sample in a buffered aqueous solution of 1.5-3.5%, preferably 2-3%, more preferably 2.2-2.75%, even more preferably 2.5% reducing agent; d) repeating step b); e) repeating step a); f) repeating step b); g) washing the sample in water; h) incubating the sample in an aqueous solution of 2.5-5.5%, preferably 3-5%, more preferably 3.5-4.5%, even more preferably 4% of a stain amplifier, preferably 1,2,3-trihydroxybenzene; i) repeating step g); j) repeating step a), wherein said heavy metal compound is dissolved in an aqueous solution. k) repeating step g); l) Incubating the sample in an aqueous solution of 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4% of a heavy metal compound. wherein said heavy metal compound is different from that in step a), m) further incubating the sample of step l), where the temperature is increased to at least 35° C.; and n) repeating step g).

[0073] staining The present invention relates to large-volume en bloc staining of biological tissue samples for connectomics analysis. The en bloc staining method for connectomics aims to label membranes with heavy metals uniformly and with high contrast without loss of staining intensity due to penetration depth or destruction of the sample. Unless otherwise specified, the concentrations of reagents used herein refer to weight / volume percent concentrations (w / v). In the context of the present invention, the term "room temperature (RT)" refers to a temperature between 20°C and 26°C. Unless otherwise specified, the term "repeat" herein generally refers to one repetition of the indicated operation, i.e., step.

[0074] Biological tissue samples The term "sample" as used herein refers to a material, preferably a tissue, that is stained and post-processed, i.e., dehydrated and embedded, for further EM analysis. Typically, the sample used in the method of the present invention is a biological tissue sample. Biological samples herein include any type of biological tissue sample collected from a living or dead subject, including biopsy or autopsy. According to the present invention, the biological tissue sample is preferably collected from a mammal.

[0075] In the context of the present invention, biological tissue samples containing neurons are preferred. The neural tissue of interest may be structures with a diameter of several hundred micrometers, such as the barrels of the mouse S1 cortex, the subnuclei of the thalamus, or the orientation-selective columns of the visual cortex.

[0076] Neural tissue according to the present invention is preferably selected from the group consisting of the whole brain, brain hemispheres, cortical and / or subcortical volumes, spinal cord, and peripheral tissues including nerve endings, etc. Particularly preferred embodiments include the cortex, such as the somatosensory cortex, the parietal cortex, etc.

[0077] Fixed The method of the present invention involves the use of fixed biological tissue samples, and any suitable means for fixing biological tissue samples known in the art, such as perfusion, immersion, etc., may be used. Those skilled in the art will understand that any fixation reagent, such as paraformaldehyde, glutaraldehyde, etc., as well as mixtures of fixation reagents known in the art, may be used in the context of the present invention. The samples used according to the present invention are fixed with a buffered aqueous solution of paraformaldehyde, glutaraldehyde, and calcium chloride. Preferably, the samples are fixed with a buffered aqueous solution of 2.5% paraformaldehyde, 1.25% glutaraldehyde, and 2 mM calcium chloride. In particular, the buffered aqueous solution used for fixation is preferably 0.08 M cacodylate buffer at pH 7.4.

[0078] In the method of the present invention, the biological tissue sample has a size of more than 1 mm. Preferably, the size of the biological tissue sample is in the range of 2-15 mm. Typical sizes are 5-15 mm or 10-15 mm. In a particularly preferred embodiment of the present invention, the biological tissue sample has a size of at least 2-3 mm, and / or 5 mm (about the size of a mouse brain hemisphere), and / or 10 mm (about the size of a whole mouse brain). When the biological sample is a human sample, the sample typically has a size of 2×3×2 mm, i.e., the human sample may have a base area of ​​2-3 mm and a thickness of up to 2 mm, preferably a thickness of 2 mm. When the human sample is a large sample from human cortical tissue, i.e., a large human cortical tissue sample, the useful sample size in the present invention is in the range of a base area of ​​2-4 mm and a thickness of 2-5 mm in plan view. Typically, the sample of the desired size used in the method of the present invention is obtained by any means suitable for cutting biological specimens, including, but not limited to, a microtome, a vibratome, a razor blade, etc. The term size herein refers to all three dimensions of a sample: length, width and height (see also Figures 1a and b).

[0079] In a preferred embodiment, the samples used in the present invention are stored in a buffered aqueous solution for at least 24 hours prior to staining.

[0080] The biological tissue sample may be stained and post-processed using the methods described herein and then either immediately subjected to analysis after processing or stored, e.g., stored for an extended period of time, prior to future analysis. For example, the biological tissue sample may be processed as described above and then stored for future analysis.

[0081] The sample container used in the method of the present invention refers to any means suitable for contacting the sample to be treated with a reagent. The sample container used herein is chemically and thermally stable, and is resistant to light when containing a light-sensitive reagent. The sample container used herein is not particularly limited, and includes commercially available containers, such as micro reaction vessels (such as Eppendorf® tubes), centrifuge tubes (such as Falcon™ tubes), glassware, etc. Larger sample sizes require larger volumes of reagents, and therefore larger sample container sizes.

[0082] buffered aqueous solution In the context of the present invention, the buffered aqueous solution is not particularly limited and may be any buffer known in the art that is suitable for treating biological samples for electron microscopy, such as phosphate buffers (Sorensen buffer, Millonig buffer, sodium phosphate buffer), sulfonic acid buffers, Sym-Collidine buffer, etc.

[0083] According to the present invention, the buffered aqueous solution is preferably a cacodylate buffer, such as sodium cacodylate, potassium cacodylate, etc. In a particularly preferred embodiment of the present invention, the buffered aqueous solution is sodium cacodylate (CaC). The concentration of the cacodylate buffer used in the present invention is 0.08-0.3M, more preferably 0.08-0.2M, even more preferably 0.08 and / or 0.15M.

[0084] The pH of the buffered aqueous solution in the context of the present invention is preferably adjusted to a value between 3 and 10, preferably between 4 and 9, more preferably between 5 and 8, even more preferably to 7.4.

[0085] The means for adjusting the pH value is not limited and any suitable means known to those skilled in the art can be used. In the context of the present invention, the pH of the sodium cacodylate buffer is adjusted using sodium hydroxide (NaOH).

[0086] The protocol steps (i.e., washing in steps g), i), k), and n) in pure water create significant osmotic stresses, which can be significant for large, fragile samples. To balance these osmotic changes, when using biological tissue samples that are sensitive to osmotic stresses, such as the cerebellum, it is useful in the present invention to perform the washing in steps g), i), k), and n) in buffered aqueous solutions. Typically, these samples can be washed with aqueous solutions including cacodylate buffer, aqueous solutions containing organic substances / molecules (including, but not limited to, sucrose, ethanol, acetone, etc.), or aqueous salt solutions including KCl, NaCl, etc.

[0087] An aqueous salt solution in the context of the present invention is preferably selected from the group consisting of 0.15 M KCl and 0.15 M NaCl.

[0088] Residuals of reduced osmium, i.e. Os(vi), present after step e) (corresponding to the second incubation step with the heavy metal solution) can adversely affect the subsequent stain amplification in step h) (corresponding to the incubation step with a stain amplification agent, e.g. pyrogallol). This is particularly true when an aqueous salt solution (e.g. KCl, NaCl) is used in step g) to balance the osmotic pressure of the biological tissue sample, which is sensitive to osmotic stress. It is therefore particularly useful to remove residual Os(vi) prior to stain amplification in order to protect the integrity of the fragile sample, and any suitable means for depositing residual Os(vi) on the sample known in the art can be used.

[0089] In the context of the present invention, Os(vi) residues can be removed by incubating the fragile sample in an aqueous solution containing a reducing agent (specified in the section "Reducing Agents" below) between the third (i.e., step f)) and fourth (i.e., step g)) washing steps with water (i.e., steps g), i), k) and n)) can then be replaced by a washing step with a buffered aqueous solution as described herein (i.e., corresponding to step f)).

[0090] In this regard, it is particularly preferred that the fragile sample is incubated for at least 24 hours in an aqueous solution containing 2.5% reducing agent (e.g., 2.5% potassium ferrocyanide) dissolved in water. Further types of reducing agents and water useful in the context of the present invention are described below.

[0091] Alternatively, residual Os(vi) can be precipitated onto the sample by temporarily adjusting the pH of the aqueous salt solution. In this regard, it is preferred to lower the pH of the aqueous salt solution to 1-2 (which will cause disproportionation of Os(vi)), preferably for at least 1-2 hours, and then return it to the initial pH value (i.e., pH 7).

[0092] The aforementioned pH adjustment can be accomplished by active adjustment, ie, changing the pH of the aqueous salt solution by addition of acid / base, or by exchanging it for the same aqueous salt solution of a different pH.

[0093] Yet another useful alternative for stabilizing fragile biological tissue samples may be to replace the second incubation step with a heavy metal solution in step e) with an incubation step with a buffered aqueous solution, such as a cacodylate buffer, or a buffered aqueous solution of the fixation reagent used herein (e.g. paraformaldehyde, glutaraldehyde dissolved in a cacodylate buffer according to the invention), or an aqueous salt solution containing KCl, NaCl, CaCl2, etc. It is preferable to use KCl, preferably at a concentration of 0.1 to 0.4 M, more preferably at a concentration of 0.15 M. In the context of the present invention, the aforementioned incubation steps may differ in time (e.g. 24 h, 48 h), temperature (e.g. 4 ° C, room temperature), etc. Furthermore, this incubation step can be repeated several times. Here, it is particularly useful to repeat this incubation step four times in succession.

[0094] In the case of human samples sensitive to osmotic stress (e.g. human samples with large blood vessels such as human cortical tissue samples), despite the above measures for osmotic balancing, it may be further useful to gradually decrease / increase the osmolarity of the incubation solution to / from the washing steps with pure water (i.e. from steps f) to g), g) to h), h) to i), i) to j), j) to k), k) to l), and l) to m)) in order to preserve the integrity of the sample. For example, considering steps f) to g), instead of changing the buffered aqueous solution to water in one step, it can be gradually changed to a solution with a decreasing osmolarity in two or more steps. The gradual change here likewise relates to a gradual decrease and a stepwise increase in the osmolarity of the incubation solution. For example, considering steps g) and h), the osmolarity of the aqueous solution of the dye amplifier can also be increased stepwise / progressively.

[0095] Heavy metal solution In the context of the present invention, to increase the density of a biological tissue sample for EM, heavy metal atoms may be introduced from solution into the tissue of interest. These heavy metal atoms attach to subcellular structures within the tissue and increase their electron dose. As a result, the subcellular structures appear dark and stand out against a light background.

[0096] Typically, heavy metals / heavy metal compounds used in accordance with the present invention include, but are not limited to, osmium tetroxide, osmic acid, uranyl acetate (UA), uranyl formate, lanthanum nitrate, phosphotungstic acid, phosphomolybdic acid, ammonium molybdate, sodium urate, lead perchlorate, erbium acetate, gadolinium acetate, methylamine tungstic acid, samarium acetate, thulium acetate, sodium silicotungstate, sodium phosphotungstate, lead hydroxide / citrate, etc. When a heavy metal solution containing lead is used in the method of the present invention, it is suggested that the incubation time is preferably extended to 24-48 hours and the solution is preferably replaced 5-10 times.

[0097] According to the present invention, water-soluble heavy metals / heavy metal compounds are preferred. In a particularly preferred embodiment of the present invention, the heavy metal compound in steps a), e) and j) is osmium tetroxide, and the heavy metal compound in step l) is uranyl acetate. In this specification, the terms "OsO4" and "osmium(viii) oxide" are used interchangeably and refer to osmium tetroxide, also known in the art as osmic acid. The water-soluble heavy metal compounds can be dissolved in either aqueous and / or buffered aqueous and / or aqueous salt solutions.

[0098] It is particularly preferred that the heavy metal compounds of steps a) and e) are dissolved in a buffered aqueous solution. Preferably, in the context of the present invention, for steps a) and e), a buffered aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% osmium tetroxide (OsO4) is used.

[0099] It is also particularly preferred that the heavy metal compounds of steps j) and l) are present as aqueous solutions. Preferably, in the context of the present invention, for step j) an aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% osmium tetroxide (OsO4) is used, and for step l) an aqueous solution of 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4% uranyl acetate (UA) is used.

[0100] In another preferred embodiment of the present invention, the heavy metal compound in steps Aa) and Ag) is osmium tetroxide and the heavy metal compound in step Ai) is uranyl acetate.

[0101] It is particularly preferred that the heavy metal compounds of steps Aa) and Ag) are dissolved in a buffered aqueous solution. Preferably, in the context of the present invention, for steps Aa) and Ag), a buffered aqueous solution of osmium tetroxide (OsO4) is used at 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2%. Alternatively, the heavy metal compounds of step Ag) can be present as an aqueous solution. Preferably, in the context of the present invention, for step Ag), a buffered aqueous solution of osmium tetroxide (OsO4) at 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% is used. It is also particularly preferred that the heavy metal compounds of step Ai) are present as an aqueous salt solution. Preferably, in the context of the present invention, for step Ai), a buffered aqueous solution of uranyl acetate (UA) at 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4% is used.

[0102] Reducing Agent To increase the film contrast and / or accelerate the contrast enhancement, a reducing agent can be used in the method of the present invention, and any reducing agent known in the art (e.g., K3Co(CN)6, K2Ru(CN)6, K4Os(CN)6) can be used. Preferably, potassium ferrocyanide (K4Fe(CN)6), potassium ferricyanide (K3Fe(CN)6), calcium ferrocyanide Ca2[Fe(CN)6], sodium ferrocyanide (Na4Fe(CN)6), etc., as well as mixtures of said reducing agents can be used in the method of the present invention. The use of a mixture of ferricyanide (Fe(iii)CN) and ferrocyanide (Fe(ii)CN) is particularly useful in the method of the present invention to titrate the background contrast when the conductivity needs to be increased. According to the present invention, potassium ferrocyanide and / or potassium ferricyanide are most preferred.

[0103] To protect background proteins after prolonged OsO4 incubation (i.e., when using samples of 5 mm and 10 mm size), steps a), b), and c) are performed below 10°C, more preferably at 2-6°C, and even more preferably at 4°C.

[0104] Alternatively, or in combination with the aforementioned measures for suppressing reducing agent membrane contrast enhancement, steps a), b), and c), preferably steps a) and b), more preferably step c), may further include calcium chloride (CaCl2). Typically, useful concentrations of CaCl2 herein include, but are not limited to, 1-10 mM, preferably 2-5 mM, more preferably 2-3 mM. In a particularly preferred embodiment, a buffered aqueous solution containing 3 mM CaCl2 is used in steps a), b), and / or c).

[0105] In the context of the present invention, "FeCN" and "K4Fe(CN)6" are used interchangeably and refer to potassium ferrocyanide. Preferably, in the context of the present invention, a buffered aqueous solution of 1.5-3.5%, preferably 2-3%, more preferably 2.2-2.75%, even more preferably 2.5% FeCN is used as the reducing agent.

[0106] water According to the present invention, the term "water" refers to purified water. Purified water refers to water that has been mechanically filtered or otherwise treated to remove impurities. Purified water in this context refers to distilled water, deionized water, autoclaved water, Nanopure™ water, Milli-Q water, etc. The use of Milli-Q water is particularly preferred.

[0107] Dye amplifiers such as pyrogallol To further enhance the staining contrast of the cellular permeability components, especially lipids, and to increase the electrical conductivity of the sample, the sample can be incubated with a stain amplifier (contrast enhancer). These stain amplifiers typically include dihydroxy, trihydroxy, and tetrahydroxy derivatives of benzene, such as 1,2,3-trihydroxybenzene (pyrogallol), 1,2,4,5-tetrahydroxybenzene, 3-methyl-1,2-benzenediol (3-methylcatechol), and 4-methyl-1,2-benzenediol (4-methylcatechol). Pyrogallol (abbreviated as "Pg" herein) is the most commonly used for this purpose and is the preferred stain amplifier herein.

[0108] In the method of the present invention, incubation with a dye amplifier, preferably 1,2,3-trihydroxybenzene, is carried out. In the context of the present invention, the terms "1,2,3-trihydroxybenzene", "pyrogallol" and "Pg" are used interchangeably. Whenever "Pg" is used in the method of the present invention, in alternative embodiments, it can be replaced with other dye amplifiers, such as 1,2,4,5-tetrahydroxybenzene, 3-methyl-1,2-benzenediol (3-methylcatechol) and 4-methyl-1,2-benzenediol (4-methylcatechol).

[0109] Preferably, in the context of the present invention, a 2.5-5.5%, preferably 3-5%, more preferably 3.5-4.5%, even more preferably 4% aqueous solution of a dye amplifier, preferably 1,2,3-trihydroxybenzene, is used in step h).

[0110] In another preferred embodiment of the present invention, the dye amplifier, preferably Pg, is prepared within less than one month, preferably less than two weeks, more preferably less than three days, even more preferably within one hour.

[0111] Incubation and washing times According to the present invention, the incubation time is long enough to allow the relevant reagents to diffuse from the outside to the center of the sample, but short enough not to cause damage at the macro- and micro-level. Furthermore, the washing time used here is long enough to remove previous reagents, thus avoiding potential undesirable chemical interactions. The incubation and / or washing time used here can vary depending on the size of the sample.

[0112] In the context of the present invention: 2~3mm The incubation time and washing time for a sample of this size are preferably 3 to 30 hours, more preferably 18 to 28 hours, and even more preferably 24 hours in step a), preferably 0.5 to 24 hours, more preferably 1 to 12 hours, and even more preferably 2 hours in step b), preferably 12 to 48 hours, more preferably 15 to 24 hours, and even more preferably 17 hours in step c), preferably 0.5 to 24 hours, more preferably 1 to 12 hours, and even more preferably 2 hours in step d), preferably 1.5 to 24 hours, more preferably 3 to 10 hours, and even more preferably 3 hours in step e), preferably 0.5 to 24 hours, more preferably 1 to 12 hours, and even more preferably 1 hour in step f), and preferably 1 to 24 hours, more preferably 1 to 17 hours in step g). in step h), the time is preferably 10 to 48 hours, more preferably 12 to 24 hours, even more preferably 17 hours; in step i), the time is preferably 1 to 24 hours, more preferably 1 to 17 hours, even more preferably 2 hours; in step j), the time is preferably 5 to 24 hours, more preferably 5 to 10 hours, even more preferably 6 hours; in step k), the time is preferably 1 to 24 hours, more preferably 1 to 17 hours, even more preferably 1 hour; in step l), the time is preferably 10 to 24 hours, more preferably 12 to 20 hours, even more preferably 17 hours; in step m), the time is preferably 1 to 6 hours, more preferably 2 to 4 hours, even more preferably 2 hours; and in step n), the time is preferably 1 to 24 hours, more preferably 1 to 17 hours, even more preferably 1 hour.

[0113] In the context of the present invention, for human samples with a sample size of 2-3 mm in each dimension (e.g. 2-3 mm in the plane (base area) and a thickness ≦2 mm), the washing step d) can be omitted, although this is not recommended. In this case, the incubation step with the buffered aqueous solution of the reducing agent would be immediately followed by an incubation step with the buffered aqueous solution of the heavy metal compound, without intermediate washing steps.

[0114] In the context of the present invention: 5mmThe incubation time and washing time for a sample of this size are preferably 40 to 90 hours, more preferably 60 to 80 hours, and even more preferably 72 hours in step a), preferably 72 to 120 hours, more preferably 80 to 100 hours, and even more preferably 96 hours in step b), preferably 48 to 96 hours, more preferably 60 to 80 hours, and even more preferably 72 hours in step c), preferably 24 to 72 hours, more preferably 40 to 60 hours, and even more preferably 48 hours in step d), preferably 24 to 72 hours, more preferably 30 to 50 hours, and even more preferably 48 hours in step e), preferably 17 to 72 hours, more preferably 20 to 40 hours, and even more preferably 24 hours in step f), and preferably 17 to 48 hours, more preferably 20 to 30 hours in step g). in step h), the time is preferably 20 to 48 hours, more preferably 20 to 30 hours, and even more preferably 24 hours; in step i), the time is preferably 17 to 48 hours, more preferably 20 to 30 hours, and even more preferably 24 hours; in step j), the time is preferably 40 to 64 hours, more preferably 40 to 50 hours, and even more preferably 48 hours; in step k), the time is preferably 17 to 48 hours, more preferably 20 to 30 hours, and even more preferably 24 hours; in step l), the time is preferably 30 to 64 hours, more preferably 40 to 50 hours, and even more preferably 48 hours; in step m), the time is preferably 1 to 6 hours, more preferably 2 to 5 hours, and even more preferably 5 hours; and in step n), the time is preferably 17 to 48 hours, more preferably 20 to 30 hours, and preferably 24 hours.

[0115] In the context of the present invention: 10mmThe incubation time and washing time for a sample of this size are preferably 70 to 120 hours, more preferably 90 to 100 hours, and even more preferably 96 hours in step a), preferably 120 to 190 hours, more preferably 144 to 170 hours, and even more preferably 96 hours in step b), preferably 68 to 96 hours, more preferably 70 to 80 hours, and even more preferably 72 hours in step c), preferably 70 to 120 hours, more preferably 80 to 100 hours, and even more preferably 72 hours in step d), preferably 24 to 72 hours, more preferably 30 to 50 hours, and even more preferably 48 hours in step e), preferably 24 to 96 hours, more preferably 48 to 80 hours, and even more preferably 72 hours in step f), and preferably 30 to 96 hours, more preferably 40 to 64 hours in step g). in step h), the time is preferably 30 to 64 hours, more preferably 40 to 50 hours, even more preferably 48 hours; in step i), the time is preferably 30 to 96 hours, more preferably 40 to 64 hours, even more preferably 48 hours; in step j), the time is preferably 72 to 120 hours, more preferably 80 to 100 hours, even more preferably 96 hours; in step k), the time is preferably 30 to 96 hours, more preferably 40 to 64 hours, even more preferably 48 hours; in step l), the time is preferably 30 to 64 hours, more preferably 40 to 50 hours, even more preferably 48 hours; in step m), the time is preferably 1 to 6 hours, more preferably 2 to 5 hours, even more preferably 5 hours; and in step n), the time is preferably 1 to 96 hours, more preferably 40 to 64 hours, even more preferably 48 hours.

[0116] In the context of the present invention, the incubation and washing times for samples derived from cerebellum and / or large human cortical tissue are preferably 40 to 90 hours, more preferably 60 to 80 hours, even more preferably 72 hours in step Aa), preferably 72 to 120 hours, more preferably 80 to 100 hours, even more preferably 96 hours in step Ab), preferably 48 to 96 hours, more preferably 60 to 80 hours, even more preferably 72 hours in step Ac), preferably 24 to 72 hours, more preferably 40 to 60 hours, even more preferably 48 hours in step Ad), and preferably 24 to 72 hours, more preferably 30 to 50 hours, even more preferably 48 hours in step Ae). in step Af), preferably 24 to 72 hours, more preferably 40 to 60 hours, even more preferably 48 hours; in step Ag), preferably 40 to 64 hours, more preferably 40 to 50 hours, even more preferably 48 hours; in step Ah), preferably 20 to 48 hours, more preferably 20 to 30 hours, even more preferably 24 hours; in step Ai), preferably 40 to 64 hours, more preferably 40 to 50 hours, even more preferably 48 hours; in step Aj), preferably 1 to 6 hours, more preferably 2 to 5 hours, even more preferably 5 hours; and in step Ak), preferably 20 to 48 hours, more preferably 20 to 30 hours, even more preferably 24 hours.

[0117] Incubation and washing temperatures According to the present invention, the incubation temperature used herein may vary depending on the size of the sample.

[0118] In the context of the present invention: 2~3mmThe incubation temperature for a sample of this size is, in step a), higher than 4°C, preferably 4 to 30°C, more preferably 15 to 25°C, even more preferably 25°C; in step c), preferably lower than 10°C, more preferably 2 to 6°C, even more preferably 4°C; in steps e), h), and j), higher than 4°C, preferably 4 to 30°C, more preferably 15 to 25°C, even more preferably 25°C; in step l), preferably lower than 10°C, more preferably 2 to 6°C, even more preferably 4°C; and in step m), preferably at least 35°C, more preferably 40 to 60°C, even more preferably 50°C.

[0119] In the context of the present invention: 5mm and 10mm The incubation temperatures for a sample of this size are preferably below 10°C, more preferably 2-6°C, even more preferably 4°C in steps a) and c), higher than 4°C, preferably 4-30°C, more preferably 15-25°C, even more preferably 25°C in steps e), h) and j), preferably below 10°C, preferably 2-6°C, even more preferably 4°C in step l), and preferably at least 35°C, more preferably 40-60°C, even more preferably 50°C in step m).

[0120] In the context of the present invention, the washing temperature does not depend on the sample size and is preferably below 10°C, more preferably between 2 and 6°C, even more preferably between 4°C, in step b) and higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C, in steps d), f), g), i), k) and m).

[0121] In the context of the present invention, the incubation temperatures for samples from cerebellum and / or large human cortical tissue are preferably below 10°C, more preferably between 2 and 6°C, even more preferably between 4°C, for steps Aa), Ab), Ac) and Ai), and higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably between 25°C, and for step Ad), Ae), Af), Ag), Ah) and Ak), and preferably at least 35°C, more preferably between 40 and 60°C, even more preferably at 50°C, for step Aj).

[0122] Solution update frequency In the context of the present invention, the incubation and washing solutions can be renewed depending on the size of the sample.

[0123] In the context of the present invention: 2~3mm The incubation solution and / or washing solution for a sample of size is preferably changed every 0.5 to 1 hour, more preferably every 0.5 to 0.75 hours, and even more preferably every 0.5 hours in step b), preferably every 12 to 48 hours, more preferably every 15 to 24 hours, and even more preferably every 24 hours in step c), preferably every 0.5 to 1 hour, more preferably every 0.5 to 0.75 hours, and even more preferably every 0.5 hours in steps d) and f), and preferably every 0.5 to 1 hour, more preferably every 0.5 to 0.75 hours, and even more preferably every 0.5 hours in steps g), i), k), and n).

[0124] In the context of the present invention: 5mm and 10mmThe incubation solution and / or washing solution for a sample of size is preferably changed every 4 to 17 hours, more preferably every 4 to 8 hours, even more preferably every 4 hours in step b), preferably every 8 to 30 hours, more preferably every 12 to 28 hours, even more preferably every 24 hours in step c), preferably every 4 to 24 hours, more preferably every 4 to 17 hours, even more preferably every 4 hours in step d), preferably every 4 to 24 hours, more preferably every 4 to 17 hours, even more preferably every 4 hours in step f), preferably every 4 to 24 hours, more preferably every 4 to 17 hours, even more preferably every 4 hours in steps g), i), k), and n), preferably every 4 to 17 hours, more preferably every 4 to 10 hours, even more preferably every 4 hours.

[0125] In the context of the present invention, the incubation solutions and / or washing solutions for samples derived from cerebellum and / or large human cortical tissue are preferably changed every 4 to 17 hours, more preferably every 4 to 8 hours, even more preferably every 4 hours in steps Ab), Ad), Af), Ah) and Ak), and preferably changed every 8 to 30 hours, more preferably every 12 to 28 hours, even more preferably every 24 hours in step Ac).

[0126] In a second aspect, the present invention relates to the post-processing of a stained biological sample. According to the present invention, the post-processing comprises dehydration and embedding of the biological tissue sample.

[0127] dehydration In the context of the present invention, after staining, the biological tissue sample is dehydrated using a graded ethanol series. Any means suitable for dehydrating biological tissue samples is suitable for the method of the present invention.

[0128] Typically, the biological tissue samples of the present invention are dehydrated through an ascending alcohol series. In particular, in the context of the present invention, the stained biological tissue sample is then placed in aqueous solutions of 25%, 50%, and 75% ethanol, as well as 100% ethanol and 100% acetone to prevent distortion of the sample due to rapid dehydration.

[0129] The dehydration procedure and temperature can vary depending on the size of the sample.

[0130] In the context of the present invention: 2~3mm The samples of size 100 μl can then be incubated in 50% and 75% aqueous solutions and in 100% ethanol. According to the invention, the incubation step in 100% ethanol is carried out at 25° C. and for the lower ethanol concentrations at 4° C.

[0131] Herein, each incubation step is preferably carried out for 0.5 to 1 hour, more preferably 0.75 hours. As a final dehydration step, the sample can be incubated in 100% acetone at 25° C. for 2 to 3 hours. Preferably, the acetone can be changed every 0.5 to 0.75 hours.

[0132] In the context of the present invention: 5mm and 10mm Samples of this size can then be incubated in 25%, 50%, and 75% aqueous solutions as well as 100% ethanol. According to the present invention, the incubation step in 100% ethanol is carried out at 25° C. and / or 4° C., with lower ethanol concentrations being carried out at 4° C. For example, when a 10 mm sample (e.g., a whole mouse brain) is to be fully embedded using the methods described herein, the incubation in 100% ethanol can be carried out at 4° C. 5mm In the case of such a sample, each incubation step may be carried out for preferably 8 to 17 hours, more preferably 8 hours. 10mmFor samples of this type, each incubation step may be carried out for 12 to 24 hours, preferably 17 to 24 hours, more preferably 24 hours. It will be understood that the incubation times for aqueous solutions having different ethanol concentrations do not necessarily have to be the same and therefore may vary depending on the ethanol concentration. For example, 10mm When a sample (e.g., a whole mouse brain) is to be fully embedded using the methods described herein, the sample can be incubated in 25% ethanol in water for 12-24 hours, preferably 12 hours, although incubations in 50% and 75% ethanol in water as well as 100% ethanol can be performed for 24 hours. As a final dehydration step, the sample can be incubated in 100% acetone at 25°C for 32 hours. 5mm For samples, acetone was replaced every 8 to 17 hours. 10mm It is preferable to change the acetone every 17-24 hours for samples. For example, when fully embedding a 10 mm sample (e.g., a whole mouse brain) using the methods described herein, the sample can be incubated in 100% acetone at 25 °C or 4 °C for 3 days.

[0133] In the context of the present invention, samples from the cerebellum and / or large human cortical tissue samples can then be incubated in 25%, 50% and 75% aqueous solutions as well as 100% ethanol. According to the present invention, the aforementioned incubation steps can be carried out at 25° C., preferably for 8-24 hours. As a final dehydration step, the samples can be incubated in 100% acetone at 25° C. for 32-48 hours. Preferably, in the case of samples from the cerebellum and / or large human cortical tissue samples, the acetone can be changed every 8-12 hours.

[0134] embedding Generally, the biological tissue sample of the present invention is embedded by using a low viscosity epoxy resin embedding method after dehydration.

[0135] In the context of the present invention, the term "resin infiltration" refers to treating tissue with a resin that permeates throughout the tissue down to the molecular level, then hardens and finally turns into a solid, hardening the sample.

[0136] According to the present invention, the term "embedding" refers to placing the infiltrated biological tissue sample into a mold, in which the sample is usually surrounded by the same infiltrating resin. The mold is then hardened to form an embedded block. The embedding reagent thus serves to provide a solid support and facilitate the subsequent sectioning process.

[0137] The embedding medium used herein is preferably an epoxy resin, including, but not limited to, commercially available epoxy resins such as Epon 812 replacement resin, resins formulated according to Spur, and the like.

[0138] In a particularly useful embodiment of the invention, the resin by Sparre (Sparre Resin, 10 g) consists of 3,4-epoxycyclohexylmethyl 3,4-epoxycyclohexane (ERL 4221, 4.1 g), diglycidyl ether of polypropylene glycol (DER 736, 0.95 g), nonenylsuccinic anhydride (NSA, 5.9 g), and dimethylaminoethanol (DMAE, preferably 11-113 μl, more preferably 55-113 μl, even more preferably 113 μl).

[0139] In another exemplary embodiment of the present invention, the Epon 821 replacement resin (Epon 812, 10 g) is composed of triglycidyl ether of glycerol (5.9 g), methyl nadic anhydride (MNA, 3.7 g), dodecenyl succinic anhydride (DDSA, 2.25 g), 2,4,6-tris(dimethylaminomethyl)phenol (DMP, 20-205 μl, more preferably 100-205 μl, even more preferably 205 μl).

[0140] To achieve uniform resin infiltration, the method of the present invention involves the use of a low viscosity resin infiltration method in which the concentration of the resin is gradually increased. The resin can be diluted by any suitable means known in the art, including organic solvents such as alcohols, ketones, etc. In a preferred embodiment of the present invention, the resin is dissolved in acetone.

[0141] The dehydration operation can be varied depending on the size of the sample. Usually, the low-viscosity epoxy resin infiltration method of the present invention is preferably carried out at less than 10°C, more preferably at 2 to 6°C, and even more preferably at 4°C.

[0142] According to the present invention, 2~3mm The samples of size can then be incubated in 25%, 50% and 75% Spurr resin in acetone. Here the aforementioned incubation step is preferably carried out for 8-16 hours, more preferably 8-10 hours, even more preferably 8 hours. As a further embedding step the samples can be incubated in 100% Spurr resin. Here the incubation in 100% Spurr resin can be carried out for preferably 2-3 days, more preferably 2 days. Furthermore, it is preferred that the pure resin is replaced every 8-17 hours, more preferably every 8 hours. In the context of the present invention, the temperature can be further increased and the samples further incubated in 100% Spurr resin.

[0143] In another embodiment of the present invention, 2~3mm The samples of size 1000 μm may be subsequently incubated with 12.5%, 25%, 37.5%, 50%, 62.5%, 75%, and 87.5% Epon 812 resin in acetone, wherein the aforementioned incubation step is preferably carried out for 4 to 16 hours, more preferably 4 to 10 hours.

[0144] It is particularly preferred that each incubation step between 12.5% ​​and 50% Epon 812 is carried out for 4 hours, and each step between 62.5% and 87.5% Epon 812 is carried out for 8 hours.

[0145] As a further embedding step, the sample can be incubated in 90% and / or 95% Epon 812 resin and 100% Epon 812 resin in acetone, whereby the aforementioned incubation steps are preferably carried out for 2 to 3 days. In particular, it is preferred that the incubation in 90% and / or 95% Epon 812 is carried out for 3 days and the incubation in 100% Epon 812 is carried out for 3 days.

[0146] According to the present invention, the 5 mm sized samples can then be incubated in 25%, 50% and 75% Sparre resin in acetone. As used herein, the aforementioned incubation steps are preferably carried out for 17-48 hours, more preferably 20-30 hours, and even more preferably 24 hours.

[0147] As a further embedding step, the sample can then be incubated in 90%, 95% Spurr resin and 100% Spurr resin in acetone. Herein, the aforementioned incubation steps are preferably carried out for 2-3 days. In particular, it is preferred that the incubation in 90% Spurr resin is carried out for 2 days and the incubation in 95% and 100% Spurr resin is carried out for 3 days. Furthermore, it is preferred that the pure resin is replaced every 8-17 hours, more preferably every 8 hours.

[0148] According to the present invention, 10mmThe size sample can then be incubated in 25%, 50% and 75% Spurr resin in acetone. Herein, the aforementioned incubation step is preferably carried out for 8 to 16 hours, more preferably for 8 to 10 hours, even more preferably for 8 hours. As a further embedding step, the sample can then be incubated in 90%, 95% and 100% Spurr resin in acetone. Herein, the aforementioned incubation step is preferably carried out for 3 to 5 days. In particular, it is preferred that incubation in 90% Spurr resin is carried out for 3 days, in 95% for 5 days and in 100% Spurr resin for 4 days. Furthermore, it is preferred that the pure resin is replaced every 8 to 17 hours, more preferably every 8 hours.

[0149] Alternatively, for whole-volume embedding, 10mm The sized samples can then be incubated in 25%, 50%, 75%, and 90% Spurr resin in acetone. The aforementioned incubation steps are preferably carried out for 2-4 days, more preferably 2-3 days, and even more preferably 3 days. As a further embedding step, the samples can then be incubated in 95% Spurr resin in acetone for 4-7 days, preferably 4 days. The samples can then be incubated in 100% Spurr resin for 6-8 days, more preferably 6-7 days, and even more preferably 6 days. Furthermore, it is preferred that the 95% Spurr resin and the pure resin are replaced every 1-2 days with freshly prepared 95% Spurr resin and freshly prepared pure resin, respectively.

[0150] According to the present invention, samples from the cerebellum and / or large human cortical tissue samples can then be incubated in 25%, 50%, 75%, 90% and 95% Spurr resin and 100% Spurr resin in acetone.

[0151] Herein, the aforementioned incubation step is preferably carried out for 24 to 36 hours. As a further embedding step, the sample can then be incubated in 100% Spurr's resin. Herein, the aforementioned incubation step is preferably carried out for 3 to 4 days. Furthermore, it is preferred that the pure resin is replaced every 8 to 17 hours, more preferably every 8 hours.

[0152] In the context of the present invention, as a final embedding step, the temperature can be further increased and the sample further incubated in pure resin, preferably for 1-3 days, more preferably for 3 days. Preferably, the temperature can be increased to 50-80° C., more preferably to 65-75° C., even more preferably to 60-70° C. When using Spurr resin, it is particularly preferred that the temperature is increased to 70° C., and when using Epon 812 resin, it is particularly preferred that the temperature is increased to 60° C.

[0153] In a third aspect, the invention relates to the analysis of stained and post-processed biological samples. According to the invention, EM analysis is preferred.

[0154] analysis The EM analysis is not limited to a particular method, and preferably volumetric EM, more preferably high-throughput volumetric EM, is used for connectomics analysis / high-density neural circuit reconstruction in the context of the present invention.

[0155] Typically, image data required for high-resolution connectomics can be obtained using a transmission electron microscope (TEM), preferably a scanning electron microscope (SEM), and / or a section-based SEM (SBEM), etc. Typically, the biological tissue sample of the present invention is cut into sections, preferably of 20-200 nm, more preferably of 30-40 nm, even more preferably of 35 nm and / or 38 nm, and further imaged using a high-throughput SEM, preferably a multi-SEM.

[0156] In one embodiment, the present invention is used for the pathological examination of mammalian, preferably human, nervous tissue samples.Pathological examination based on the method of the present invention can be useful for the study of neurological diseases, including but not limited to Alzheimer's disease, Parkinson's disease, and autism.In addition, the method of the present invention can also be used to evaluate the effectiveness of therapeutic measures such as drug therapy in the aforementioned neurological diseases.

[0157] In a preferred embodiment, the present invention relates to a method for staining a biological tissue sample having a sample size of 2-3 mm, the method comprising the steps of: a) incubating the sample in 2% (w / v) osmium tetroxide (OsO4) dissolved in 0.15 M sodium cacodylate (CaC) buffer (pH 7.4), wherein said incubation is carried out at 25° C. for 24 hours; b) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein said washing is carried out for 2 hours at 4° C. and wherein the buffer is changed every 0.5 hours; c) incubating the sample in 2.5% (w / v) potassium ferrocyanide (KFe(CN)) dissolved in 0.15 M CaC buffer (pH 7.4), wherein said incubation is carried out at 4° C. for 17 hours; d) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein said washing is carried out for 2 hours at 25° C. and wherein the buffer is changed every 0.5 hours; e) incubating the sample in 2% OsO4 (w / v) dissolved in 0.15 M CaC buffer (pH 7.4), wherein the incubation is carried out at 25° C. for 3 hours; f) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein the wash is carried out at 25° C. for 1 hour; and wherein the buffer is changed every 0.5 hours; g) washing the sample with purified water, wherein the wash is carried out at 25° C. for 2 hours; and wherein the water is changed every 0.5 hours; h) incubating the sample in 4% (w / v) 1,2,3-trihydroxybenzene dissolved in purified water, wherein said incubation is carried out at 25° C. for 17 hours; i) washing the sample with purified water, wherein the wash is carried out at 25° C. for 2 hours; and wherein the water is changed every 0.5 hours; j) incubating the sample in 2% (w / v) OsO4 dissolved in purified water, wherein said incubation is carried out at 25° C. for 6 hours; k) washing the sample with purified water, wherein the wash is carried out at 25° C. for 1 hour; and wherein the water is changed every 0.5 hours; l) incubating the sample in an aqueous solution of 4% (w / v) uranyl acetate (UA), wherein said incubation is carried out at 4° C. for 17 hours; m) further incubating the sample of step l), wherein said incubation is carried out at 50° C. for 2 hours; and, n) washing the sample with purified water, wherein the wash is carried out at 25° C. for 1 hour; And, wherein the water is changed every 0.5 hours.

[0158] In another preferred embodiment, the present invention relates to a method for staining a biological tissue sample having a sample size of 5 mm, the method comprising the steps of: a) incubating the sample in 2% (w / v) osmium tetroxide (OsO4) dissolved in 0.15 M sodium cacodylate (CaC) buffer (pH 7.4), wherein said incubation is carried out at 4° C. for 72 hours; b) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein said washing is carried out for 96 hours at 4° C. and wherein the buffer is changed every 4 hours; c) incubating the sample in 2.5% (w / v) potassium ferrocyanide (KFe(CN)) dissolved in 0.15 M CaC buffer (pH 7.4), wherein the incubation is carried out at 4° C. for 72 hours; and wherein the FeCN is replaced every 24 hours; d) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein said washing is carried out for 48 hours at 25°C; and wherein the buffer is changed every 4 hours; e) incubating the sample in 2% OsO4 (w / v) dissolved in 0.15 M CaC buffer, wherein the incubation is carried out at 25° C. for 48 hours; f) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein said washing is carried out for 24 hours at 25°C; and wherein the buffer is changed every 4 hours; g) washing the sample with purified water, wherein said washing is carried out for 24 hours at 25°C; and wherein the water is changed every 4 hours; h) incubating the sample in 4% (w / v) 1,2,3-trihydroxybenzene dissolved in purified water, wherein said incubation is carried out at 25° C. for 24 hours; i) washing the sample with purified water, wherein said washing is carried out for 24 hours at 25°C; and wherein the water is changed every 4 hours; j) incubating the sample in 2% (w / v) OsO4 dissolved in purified water, wherein said incubation is carried out at 25° C. for 48 hours; k) washing the sample with purified water, wherein said washing is carried out for 24 hours at 25°C; and wherein the water is changed every 4 hours; l) incubating the sample in an aqueous solution of 4% (w / v) uranyl acetate (UA), wherein said incubation is carried out at 4° C. for 48 hours; m) further incubating the sample of step l), wherein said incubation is carried out at 50° C. for 5 hours; and, n) washing the sample with purified water, wherein said washing is carried out for 24 hours at 25°C; and wherein the water is changed every 4 hours.

[0159] In yet another preferred embodiment, the present invention relates to a method for staining a biological tissue sample having a sample size of 10 mm, the method comprising the steps of: a) incubating the sample in 2% (w / v) osmium tetroxide (OsO4) dissolved in 0.15 M sodium cacodylate (CaC) buffer (pH 7.4), wherein said incubation is carried out at 4° C. for 96 hours; b) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein the wash is carried out at 4° C. for 168 hours; and wherein the buffer is changed every 4 hours; c) incubating the sample in 2.5% (w / v) potassium ferrocyanide (KFe(CN)) dissolved in 0.15 M CaC buffer (pH 7.4), wherein the incubation is carried out at 4° C. for 72 hours; and wherein the FeCN is replaced every 24 hours; d) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein the wash is carried out at 25° C. for 96 hours; and wherein the buffer is changed every 4 hours; e) incubating the sample in 2% OsO4 (w / v) dissolved in 0.15 M CaC buffer (pH 7.4), wherein the incubation is carried out at 25° C. for 48 hours; f) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein said washing is carried out at 25° C. for 72 hours; and wherein the buffer is changed every 4 hours; g) washing the sample with purified water, wherein said washing is carried out for 48 hours at 25°C; and wherein the water is changed every 4 hours; h) incubating the sample in 4% (w / v) 1,2,3-trihydroxybenzene dissolved in purified water, wherein said incubation is carried out at 25° C. for 24 hours; i) washing the sample with purified water, wherein said washing is carried out for 48 hours at 25°C; and wherein the water is changed every 4 hours; j) incubating the sample in 2% (w / v) OsO4 dissolved in purified water, wherein said incubation is carried out at 25° C. for 96 hours; k) washing the sample with purified water, The wash was carried out at 25°C for 48 hours. and wherein the water is changed every 4 hours; l) incubating the sample in an aqueous solution of 4% (w / v) uranyl acetate (UA), wherein said incubation is carried out at 4° C. for 48 hours; m) further incubating the sample of step l), wherein said incubation is carried out at 50° C. for 5 hours; and, n) washing the sample with purified water, wherein said washing is carried out for 48 hours at 25°C; And here the water is changed every 4 hours.

[0160] In another preferred embodiment, the present invention further relates to a method for post-processing the biological tissue sample obtained in step n), comprising the steps of: o) dehydrating the sample through a graded ethanol series, Here, the samples were then incubated in aqueous solutions of 50%, 75% ethanol, and 100% ethanol at 25°C; and wherein each incubation step is carried out for 0.5 to 1 hour; p) incubating the sample in 100% acetone at 25° C. Here, the sample is incubated for 2-3 hours, and wherein the acetone is replaced every 0.5 to 0.75 hours. q1) embedding the sample by a low-viscosity epoxy resin infiltration method, Here, samples were then incubated in 25%, 50%, and 75% Sparre resin in acetone at 4°C; and wherein each incubation step is carried out for 8 to 17 hours; or q2) embedding the sample in a low-viscosity epoxy resin by infiltration; Here, the samples were then incubated in 12.5%, 25%, 37.5%, 50%, 62.5%, 75%, and 87.5% Epon 812 resin in acetone at 4°C, and, wherein each incubation step from 12.5% ​​to 50% is carried out for 4 to 17 hours; and wherein each incubation step from 62.5% to 87.5% is carried out for 8 to 17 hours; r1) incubating the sample of step q1) in 100% Sparre resin at 4° C., Here the samples are incubated for 2 days, and wherein the resin is changed every 8 to 17 hours; or r2) incubating the samples of step q2) in 90% Epon 812 resin and 100% Epon 812 resin in acetone at 4° C., Here, samples were incubated at 90% for 3 days and at 100% for 2 days. and where the resin is replaced every 8 to 17 hours. s) further incubating the sample of step r1), wherein the incubation is carried out at 70° C. for 1 to 3 days; or Further incubating the sample of step r2), wherein the incubation is carried out at 60° C. for 1 to 3 days; Here, the sample size is 2 to 3 mm.

[0161] In another preferred embodiment, the present invention further relates to a method for post-processing the biological tissue sample obtained in step n), comprising the steps of: o) dehydrating the sample through a graded ethanol series, Here, the samples were then incubated in 25%, 50%, 75%, and 100% ethanol at 25° C. and wherein each incubation step is carried out for 8 to 17 hours; p) incubating the sample in 100% acetone at 25° C. Here, the samples were incubated for 32 hours, and, where the acetone is changed every 8-17 hours; q) embedding the sample in a low-viscosity epoxy resin by infiltration, Here, samples were then incubated in 25%, 50%, and 75% Sparre resin in acetone at 4°C; and, wherein each incubation step is carried out for 24 hours; r) incubating the samples in 90%, 95% and 100% Sparre resin in acetone at 4° C. Here, samples were incubated for 2 days at 90%, 3 days at 95%, and 3 days at 100%, and where the resin is replaced every 8 to 17 hours. s) further incubating the sample of step r), The incubation step includes a step of performing the incubation at 70° C. for 1 to 3 days. Here the sample size is 5 mm.

[0162] In another preferred embodiment, the present invention further relates to a method for post-processing the biological tissue sample obtained in step n), comprising the steps of: o) dehydrating the sample through a graded ethanol series, Here, the samples were then incubated in 25%, 50%, 75%, and 100% ethanol at 25° C. and, wherein each incubation step is carried out for 24 hours; p) incubating the sample in 100% acetone at 25° C. Here the samples are incubated for 32 hours, and where the acetone is replaced every 8 to 17 hours. q) embedding the sample in a low-viscosity epoxy resin by infiltration, Here, samples were then incubated in 25%, 50%, and 75% Sparre resin in acetone at 4°C; and, wherein each incubation step is carried out for 24 hours; r) incubating the samples in 90%, 95% and 100% Sparre resin in acetone at 4° C. Here, samples were incubated at 90% for 3 days, 95% for 5 days, and 100% for 4 days. and where the resin is replaced every 8 to 17 hours. s) further incubating the sample of step r), wherein the incubation is carried out at 70° C. for 1 to 3 days; Here the sample size is 10 mm.

[0163] In another preferred embodiment, the present invention further relates to a method for post-processing the biological tissue sample obtained in step n), in particular a whole-volume embedding method, comprising the steps of: o) dehydrating the sample through a graded ethanol series, Here, the samples were then incubated in 25%, 50%, 75%, and 100% ethanol at 4°C, wherein the first incubation step in an aqueous solution of 25% ethanol is carried out for 12 hours, and the remaining steps are carried out for 24 hours; p) incubating the sample in 100% acetone at 25° C. Here the samples are incubated for 3 days, and wherein the acetone is replaced every 24 hours. q) embedding the sample in a low-viscosity epoxy resin by infiltration, Here, samples were then incubated in 25%, 50%, and 75% Sparre resin in acetone at 4°C; wherein the samples are incubated in 25% Spurr resin for 2 days, 50% for 2 days, and 75% for 3 days; r) incubating the samples in 90%, 95% and 100% Sparre resin in acetone at 4° C. Here, samples were incubated in 90% Spurr resin for 2 days, 95% for 4 days, and 100% for 6 days. and wherein 95% and 100% of the resin is replaced every 2 days; s) further incubating the sample of step r), wherein the incubation is carried out at 70° C. for 1 to 3 days; Here the sample size is 10 mm.

[0164] In a preferred embodiment, the present invention relates to a method for staining biological tissue samples, in particular human samples, with a sample size of 2-3 mm in each dimension (e.g., 2-3 mm in the planar (base) region and ≦2 mm in thickness), comprising the steps of: a) incubating the sample in 2% (w / v) osmium tetroxide (OsO4) dissolved in 0.15 M sodium cacodylate (CaC) buffer (pH 7.4), wherein said incubation is carried out at 25° C. for 22 hours; b) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein the wash is carried out for 2 hours at 4° C. and wherein the buffer is changed every 20 minutes; c) incubating the sample in 2.5% (w / v) potassium ferrocyanide (KFe(CN)) dissolved in 0.15 M CaC buffer (pH 7.4), wherein said incubation is carried out at 4° C. for 22 hours; d) incubating the sample in 2% (w / v) OsO4 dissolved in 0.15 M CaC buffer (pH 7.4), wherein said washing is carried out at 25° C. for 6 hours; e) washing the sample with 0.15 M CaC buffer (pH 7.4), wherein said washing is carried out at 25° C. for 0.5 hours; f) washing the sample with purified water, wherein the wash is carried out at 25° C. for 1 hour; and wherein the water is changed every 20 minutes; g) incubating the sample in 4% (w / v) 1,2,3-trihydroxybenzene dissolved in purified water, wherein the incubation is carried out at 25° C. for 18 hours; and wherein the 1,2,3-trihydroxybenzene dissolved in purified water is replaced once after 2 hours of incubation; h) washing the sample with purified water, wherein the wash is carried out at 25° C. for 1 hour; and wherein the water is changed every 20 minutes; i) incubating the sample in 2% (w / v) OsO4 dissolved in purified water, wherein said incubation is carried out at 25° C. for 6 hours; j) washing the sample with purified water, wherein the wash is carried out at 25° C. for 1 hour; and wherein the water is changed every 0.5 hours; k) incubating the sample in an aqueous solution of 4% (w / v) uranyl acetate (UA), wherein said incubation is carried out at 4° C. for 14 hours; l) further incubating the sample of step k), wherein said incubation is carried out at 50° C. for 2 hours; and, m) washing the sample with purified water, wherein the wash is carried out at 25° C. for 1 hour; And here the water is changed every 20 minutes.

[0165] In another preferred embodiment, the present invention further relates to a method for post-processing the sample obtained in step m), comprising the steps of: n) dehydrating the sample through a graded ethanol series, where the samples were then incubated in aqueous solutions of 20%, 40%, 60%, and 80% ethanol at 4° C., and in aqueous solutions of 100% ethanol at 25° C.; and, wherein each incubation step is carried out for 0.5 to 1 hour; o) incubating the sample in 100% acetone at 25° C. The sample is then incubated for 2-3 hours. and wherein the acetone is replaced every 0.5 to 0.75 hours; p) embedding the sample by low-viscosity epoxy resin infiltration, Here the samples were then incubated in 12.5%, 25%, 37.5%, 50%, 62.5%, 75%, and 87.5% Epon 812 resin in acetone at 4°C, Here, each incubation step from 12.5% ​​to 50% was carried out for 4 to 13 hours. and, wherein each incubation step from 62.5% to 87.5% is carried out for 8 to 19 hours; q) incubating the samples of step q2) in 95% Epon 812 resin and 100% Epon 812 resin in acetone at 4° C., Here, samples were incubated for 3 days at 95% and 2 days at 100%, and wherein the resin is changed every 8 to 19 hours; r) incubating the sample in 100% Epon 812 resin, wherein the incubation is carried out at 60° C. for 3 days; Here, the sample has a size of 2-3 mm in each dimension (eg, 2-3 mm in the plane (base region) and a thickness ≦2 mm), and is in particular a human sample.

[0166] In another preferred embodiment, the present invention relates to a method for staining a biological tissue sample, in particular a sample derived from cerebellar and / or human cortical tissue, comprising the steps of: Aa) incubating the sample in 2% (w / v) osmium tetroxide (OsO4) dissolved in 0.15 M sodium cacodylate (CaC) buffer (pH 7.4), wherein said incubation is carried out at 4° C. for 72 hours; Ab) washing the sample with 0.15M CaC buffer (pH 7.4); wherein the wash is carried out at 4° C. for 96 hours; and wherein the buffer is changed every 4 hours; Ac) incubating the sample in 2.5% (w / v) potassium ferrocyanide (KFe(CN)) dissolved in 0.15 M CaC buffer (pH 7.4); wherein the incubation is carried out at 4° C. for 72 hours; and wherein the FeCN is replaced every 24 hours; Ad) incubating the sample in 0.15 M CaC buffer (pH 7.4) or 0.15 M KCl, wherein said washing is carried out for 48 hours at 25°C; and wherein the buffer is changed every 4 hours; Ae) incubating the sample in 4% (w / v) 1,2,3-trihydroxybenzene dissolved in purified water, wherein said incubation is carried out at 25° C. for 24 hours; Af) repeating step Ad); Ag) incubating the sample in 2% (w / v) OsO4 dissolved in purified water or 2% (w / v) OsO4 dissolved in 0.15 M CaC buffer (pH 7.4), wherein said incubation is carried out at 25° C. for 24 hours; Ah) washing the sample with 0.15M KCl or 0.15M NaCl, wherein said washing is carried out for 24 hours at 25°C; and wherein KCl or NaCl is replaced every 4 hours; Ai) incubating the sample in 4% (w / v) uranyl acetate (UA) in 0.15M KCl or 0.15M NaCl, wherein said incubation is carried out at 4° C. for 48 hours; Aj) further incubating the sample of step Ai), wherein said incubation is carried out at 50° C. for 5 hours; and Ak) repeating step Ah).

[0167] In another preferred embodiment, the present invention further relates to a method for post-processing the biological tissue sample obtained in step Ak), said method comprising the steps of: Al) dehydrating the sample through a graded ethanol series, Here, the samples were then incubated in aqueous solutions of 25%, 50%, 75%, and 100% ethanol at 25° C. and, wherein each incubation step is carried out for 8 to 24 hours; Am) incubating the sample in 100% acetone at 25° C., wherein the sample is incubated for 32 to 48 hours; and, wherein the acetone is changed every 8-12 hours; An) embedding the sample by a low-viscosity epoxy resin infiltration method, Here, samples were then incubated in 25%, 50%, 75%, 90%, 95%, and 100% Spurr resin in acetone at 4°C, and, wherein each incubation step is carried out for 24 to 36 hours; Ao) incubating the sample of step q1) in 100% Spurr resin at 4° C., The samples were incubated for 3-4 days. and wherein the resin is replaced every 8 to 17 hours; and Ap) further incubating the sample of step Ao), Incubation is a process carried out at 70°C for 1 to 3 days.

[0168] The invention will now be further described with the aid of the following non-limiting examples. EXAMPLES

[0169] Example 1 - General Method Animal testing All experimental procedures were approved by the local Animal Care and Use Committee and conformed to the law on animal experiments issued by the German Federal Government.

[0170] Adult C57BL6 / J mice (male / female, P30-P90) were treated with analgesics (0.1 mg / kg buprenorphine (CP-Pharma) and 100 mg / kg metamizole (WDT)) 30 min prior to isoflurane anesthesia (Harvard Apparatus, 5% in O2 at initialization, 2–3% at maintenance, O2 flow rate 1 l / min). After anesthesia, animals were perfused transcardially (Harvard Apparatus, flow rate 10 ml / min) using 15 ml of sodium cacodylate buffer (0.15 M, pH 7.4, Sigma-Aldrich) and then administered 30 ml of fixative containing 2.5% paraformaldehyde (Sigma-Aldrich), 1.25% glutaraldehyde (Serva), and 2 mM calcium chloride (Sigma-Aldrich) in 0.08 M sodium cacodylate buffer (osmolality approximately 700–800 mmol / kg, pH 7.4). The time from the start of perfusion to diaphragm incision was less than 30 seconds. After perfusion, animals were decapitated, the skull was opened with care to avoid mechanical damage to the brain, and the brain was postfixed in situ for 12–96 h at 4 °C before being removed from the skull. For 2 mm samples, brains were cut into 2 mm thick coronal sections in 0.15 M cacodylate buffer using a vibratome (Leica VT1200). Then, samples were extracted from the dorsal cortex, ventral cortex, and subcortical regions using a 2 mm diameter biopsy punch (KAI Medical, Honolulu, USA) (Figure 3j). Samples were then stored in 0.15 M cacodylate buffer at 4 °C for 8–24 h before staining.

[0171] For hemisphere samples, the brains were cut along the midline with a razor blade (Wilkinson). The hemispheres were stored in 0.15 M cacodylate buffer at 4° C. for 24 hours before staining.

[0172] Human samples Human brain tissue samples (human individual H6, same as in (NPL40)) were obtained at the Neurosurgery Department of the Klinikum Rechts der Isar, Technical University of Munich, during neurosurgery procedures that were ordered for medical reasons independently of this research project. Samples were taken from access tissue (healthy brain parenchyma that had to be removed as part of the surgery and would have been discarded otherwise) before removal of the respective target lesion, with the approval of the ethical committee of the Medical Faculty of the Technical University of Munich (Ethikvotum 184 / 16S and 273 / 21 S-EB). All patients had given written informed consent.

[0173] The human "H6" sample was obtained from the inferior frontal gyrus of a 69-year-old female patient during surgical removal of a frontal mass lesion (final diagnosis: glioblastoma multiforme). After surgical removal, the tissue was collected directly into fixative stored at 4°C. The tissue was immediately sliced ​​into 2 mm thin sections in cold fixative using a vibratome. The sections were stored overnight at 4°C. Then, 2 × 3 mm sections were cut from the sections with a razor. 2 Samples were cut to size.

[0174] 2×3×2mm 3Human samples were stained according to the 2 mm protocol (Table 1) with minor modifications (Table 2). Briefly, after four 30 min rinses (4°C) with 0.15 M CaC, the following steps were performed in sequence: 2% OsO4 in 0.15 M CaC, pH 7.4, for 22 h at room temperature, four 30 min rinses with 0.15 M CaC at room temperature, 2.5% FeCN in 0.15 M CaC, pH 7.4, for 22 h at room temperature, 2% OsO4 in 0.15 M CaC, pH 7.4, for 6 h at room temperature, 0.15 M CAC for 30 min, and HO for 1 h at room temperature. 3 x 20 min rinses at room temperature, 2% pyrogallol in H2O for 18 h at room temperature, 3 x 20 min H2O rinses, 2% OsO4 in H2O for 6 h, 3 x 20 min H2O rinses, 4% UA for 14 h at 4 °C (then the temperature was switched to 50 °C for 2 h), 3 x 20 min H2O. The samples then underwent a graded ethanol dehydration step: 20% ethanol for 30 min at 4 °C, 40% ethanol for 30 min at 4 °C, 60% ethanol for 30 min at 4 °C, 80% ethanol for 30 min at 4 °C, 100% ethanol for 45 min at room temperature. For resin infiltration, after 3 x 45 min pure acetone washes, the samples were infiltrated with Epon resin graded in acetone (for 10 ml resin: 5.9 g Epon medium, 2.25 g DDSA, 3.7 g MNA, 205 μl DMP), all at 4°C: 12.5% ​​for 4 hrs, 25% for 13 hrs (overnight), 37.5% for 4 hrs, 50% for 4 hrs, 62.5% for 19 hrs (overnight), 75% for 8 hrs, 87.5% for 19 hrs (overnight), 95% for 8 hrs, 95% for 19 hrs (95% for the 1st overnight), 95% for 8 hrs, 95% for 19 hrs (95% for the 2nd overnight), 95% for 8 hrs, 95% for 19 hrs (95% for the 3rd overnight), 100% for 8 hrs, 100% for 19 hrs (100% for the 1st overnight), 100% for 8 hrs, 100% for 19 hrs (100% for the 2nd overnight); the specimens were then embedded in freshly prepared Epon resin and cured at 60°C for 3 days.

[0175] Dyeing experiment Unless otherwise stated, all staining and resin infiltration steps for 2-3 mm samples were performed in 2 ml Eppendorf tubes and for hemisphere or whole brain samples in 50 ml glass tubes at room temperature. For steps involving light-sensitive chemicals (FeCN, TCH, Pg, UA, or Ld), the tubes were covered with aluminum foil.

[0176] All chemicals used in the staining pipeline are listed in Table 3. Details of all experiments reported in the figures are given in Table 4.

[0177] For simplicity, the following terms were used to refer to the staining steps repeated in the different experiments: CaC: Sodium cacodylate buffer rinse, 0.15M pH 7.4 H2O: Water (Milli-Q®) rinse 1st Os: 1st OsO4 incubation step, 2% OsO4 in 0.15 M CaC (pH 7.4) FeCN: FeCN incubation step, 2.5% in 0.15M CaC (pH 7.4) 2nd Os: OsO4 incubation step after FeCN, 2% OsO4 in 0.15 M CaC (pH 7.4) Pg: pyrogallol incubation step, 4% in HO TCH: thiocarbohydrazide incubation step, 1% in HO 3rd Os: OsO4 incubation step after Pg, 2% in water UA: uranyl acetate incubation step, UA dissolved in water (2% or 4%) Ld: Lead aspartate incubation step, 0.66% lead nitrate in 0.03M aspartic acid (pH 5.0)

[0178] Staining of 2 mm specimens using the 1 mm protocol The 1 mm staining protocol (NPL11) with the addition of a 2 Os step was applied directly to the 2 mm samples. Samples were stained with the following steps: 1 Os 1.5 h → FeCN 1.5 h → 2 Os 1 h → CaC 0.5 h → H2O 0.5 h → TCH 1.5 h → H2O 0.5 h (twice) → 3 Os 1.5 h → H2O 0.5 h (twice) → 2% UA (17 h at 4 °C, 50 h at 50 °C) → H2O 0.5 h (twice) → Ld 2 h at 50 °C → H2O 0.5 h (twice). Samples were then incubated in a graded ethanol series from 50% (4 °C), 75% (4 °C) to 100% (45 min each step). Samples were then incubated in pure acetone three times for 45 min each time. These were then incubated with 50% Sparre resin (Sigma-Aldrich, ratio: 0.95 g ERL 4221, 5.9 g DER 736, 0.1 g NSA, 113 μl DMAE) in acetone for 6 hours. The samples were then left overnight with the caps open in the Eppendorf tubes to allow the acetone to evaporate.

[0179] The samples were then transferred to pure Sparre's resin for 6 hours, then embedded and cured at 70 °C for 1-3 days. After the resin cured, the samples were first imaged with μCT to check the uniformity of the staining. They were then trimmed to expose the center of the sample, the sample surface was smoothed, and imaged with high vacuum SEM and EDS.

[0180] Step-by-step μCT diagnosis of the main staining steps of the 1 mm protocol on a 2 mm specimen The 1 mm staining protocol (NPL11) with the addition of a 2Os step was applied to a batch of 2 mm samples as described above: 1Os 1.5 h → FeCN 1.5 h → 2Os 1 h → CaC 0.5 h → H2O 0.5 h → TCH 1.5 h → H2O 0.5 h (2 times) → 3Os 1.5 h → H2O 0.5 h (2 times) → 2% UA (17 h at 4 °C, 50 h at 50 °C) → H2O 0.5 h (2 times) → Ld 2 h at 50 °C → H2O 0.5 h (2 times). After each main step (1Os, FeCN, 2Os, 3Os, UA), two samples were removed from the staining pipeline and rinsed with CaC or H2O depending on the solvent conditions of the corresponding staining step (rinsing solutions were changed every 8 / 17 h). Once rinsing of all samples from different conditions was completed, the samples were embedded in 2% agarose in water at different tube depths in the same Eppendorf tube and stored at 4°C until the agarose hardened. The tubes containing all samples were then imaged by μCT to examine the staining gradient.

[0181] Extended FeCN incubation time for 2mm samples The 2 mm samples were stained as follows: 1Os 3 h → FeCN. Two samples were removed at each time point of 1.5, 3, 7, and 17 h during the FeCN incubation. Samples were then rinsed in CaC for 1 h and embedded in 2% agarose as described above. After agarose hardening, they were imaged by μCT to examine possible staining gradients associated with the FeCN incubation.

[0182] Interaction between OsO4 incubation time and FeCN incubation The 2 mm samples were stained under two conditions: (1) 1Os 3 h → FeCN 17 h; or (2) 1Os 24 h → FeCN 17 h. They were then rinsed sequentially with CaC (0.5 h) and HO (twice, 0.5 h each), and then they were dehydrated and embedded according to the 2 mm Sparre resin protocol. After resin embedding, they were trimmed to expose the center of the sample, the surface was smoothed, and imaged with a low-vacuum SEM to examine film contrast.

[0183] Extended TCH incubation of 2mm samples Three groups of 2 mm samples were stained and each batch was incubated with a different TCH incubation length using the following protocol: 1st Os 3 h → FeCN 17 h → 2nd Os 3 h → CaC 0.5 h → HO 0.5 h (twice) → TCH 1.5 h, or 3 h, or 5 h → HO 0.5 h (twice) → 3rd Os 3 h. They were then rinsed and embedded in 2% agarose for imaging with μCT.

[0184] Replacement of TCH with pyrogallol Three groups of 1 mm samples (each group digested with TCH-related steps) were stained using the following protocol: 1st Os 1.5 h → FeCN 1.5 h → 2nd Os 1 h → CaC 0.5 h → H2O 0.5 h → TCH 1.5 h or Pg 1.5 h or H2O 1.5 h → H2O 0.5 h (twice) → 3rd Os 1.5 h → H2O 0.5 h (twice) → 2% UA (17 h at 4 °C, 50 h at 50 °C) → H2O 0.5 h (twice) → Ld 2 h at 50 °C → H2O 0.5 h (twice). They were then dehydrated and embedded in resin according to (NPL11). After the resin cured, the samples were trimmed to expose the center, their surfaces were smoothed, and they were imaged by high-vacuum SEM and EDS. For the EDS measurements, a point measurement was selected at the neuropil at the center of the sample.

[0185] Comparison of pyrogallol incubation in HO vs. CaC Two groups of 2 mm samples (which differ in Pg incubation and water steps around Pg) were stained with the following successive incubation steps: 1st Os 24 h → FeCN 17 h → 2nd Os 3 h → CaC 0.5 h → H2O 0.5 h (twice) → Pg in water 17 h → H2O 0.5 h (twice) or CaC 0.5 h (twice) → Pg in CaC 17 h → CaC 0.5 h (twice) → 3rd Os 6 h → H2O 0.5 h (twice) → 4% UA (17 h at 4°C, 2 h at 50°C) → H2O 0.5 h (twice). Samples were then dehydrated and embedded in Spurr's resin as described above for the 2 mm samples. After the resin cured, they were trimmed to expose the center, the surface smoothed, and imaged in a high-vacuum SEM.

[0186] Long-term OsO4 incubation for 2 mm samples The 2 mm samples were stained with 2% OsO4 in CaC for 3 or 6 days. After washing in 0.15 M CaC for 0.5 h and two washes in water (0.5 h each), the samples were dehydrated and embedded according to the 2 mm Sparre's resin protocol. The samples were then trimmed to expose the center, the surfaces smoothed, and they were imaged in a low-vacuum SEM to determine the preservation of the ultrastructure.

[0187] Extended Pg and 3Os incubation of 2mm samples Three groups of 2 mm samples (these groups differ in the incubation times of the Pg and 3Os steps) were stained using the following protocol: 1Os 3 h → FeCN 17 h → 2Os 3 h → CaC 0.5 h → HO 0.5 h (twice) → Pg in water (6 h or 17 h) → HO 0.5 h (twice) → 3Os (3 h or 6 h) → HO 0.5 h (twice) → 2% UA (17 h at 4 °C, 2 h at 50 °C) → HO 0.5 h (twice). The incubation time combinations of Pg and 3Os were as follows: (1) Pg 6 h-3Os 3 h; (2) Pg 17 h-3Os 3 h; (3) Pg 17 h-3Os 6 h. After staining, the samples were dehydrated and embedded in Spurr's resin as described above for the 2 mm samples. After the resin hardened, they were trimmed to expose the center, their surfaces smoothed, and imaged in a high vacuum SEM.

[0188] UA and Ld process for 2mm sample Four groups of 2 mm samples (these groups differ in the incubation of the UA and Ld steps) were stained using the following protocol: 1st Os 3 h → FeCN 17 h → 2nd Os 3 h → CaC 0.5 h → H2O 0.5 h (twice) → Pg in water 17 h → H2O 0.5 h (twice) → 3rd Os 6 h → H2O 0.5 h (twice) → 2% or 4% UA (17 h at 4 °C, 2 h at 50 °C) → H2O 0.5 h (twice) → Ld 50 °C, 4 h or 24 h → H2O 0.5 h (twice). The combinations of UA and Ld incubations were as follows: (1) 2% UA-Ld without, (2) 2% UA-Ld for 4 h, (3) 4% UA-Ld without, and (4) 4% UA-Ld for 24 h. After staining, the samples were dehydrated and embedded in Sparre's resin as described above for the 2 mm samples. After the resin cured, the samples were imaged with a μCT, then trimmed to expose the center, the surface smoothed, and imaged with a high-vacuum SEM.

[0189] Temperature of Os incubation step Two groups of 2 mm specimens were stained according to the following procedure: (1) Os at 4°C for 7 days, or (2) Os at 4°C for 7 days, room temperature for 1 day. After staining, the specimens were dehydrated and embedded in Spurr's resin as described above for the 2 mm specimens. After the resin hardened, they were trimmed to expose the center, the surface smoothed, and imaged in a low-vacuum SEM.

[0190] Os incubation at 4 °C followed by FeCN incubation Four groups of specimens were stained according to the following procedure: (1) Os 4°C 6 days, 1 day at room temperature → FeCN 1 day at room temperature; (2) Os 4°C 6 days, 1 day at room temperature → FeCN 4°C 1 day; (3) Os 4°C 7 days → FeCN 1 day at room temperature; (4) Os 4°C 7 days → FeCN 4°C 1 day. After staining, the specimens were dehydrated and embedded in Sparre's resin as described above for the 2 mm specimens. After the resin cured, they were trimmed to expose the center, the surface smoothed, and imaged in a low-vacuum SEM.

[0191] Diffusion of Os in brain hemisphere samples at 4 °C Brain hemispheres were incubated in OsO4 at 4 °C. They were removed from the refrigerator at various time points (17 h, 24 h, and 40 h) for rapid μCT scanning (usually about 15-20 min total) and then returned to the refrigerator at 4 °C. μCT images were analyzed using Zeiss TXM3D Viewer software, where the depth of OsO4 incubation was measured in sagittal sections.

[0192] Effect of the CaC process on the staining gradient of the brain hemispheres Brain hemispheres from two groups were stained in two conditions: (1) Os 48 h → FeCN 48 h; or (2) Os 48 h → CaC 48 h → FeCN 48 h. After staining, the brain hemispheres were lightly rinsed with CaC and cut into coronal sections approximately 2 mm thick with a razor blade. The coronal sections were then dehydrated and embedded in Spurr's resin as described above for the 2 mm specimens. After the resin hardened, they were imaged in the μCT, then trimmed flat to expose the top surface, smoothed, and imaged in the low-vacuum SEM.

[0193] Effect of CaC process on FeCN diffusion rate Two groups of 2 mm samples were stained in two conditions: (1) Os 24 h → FeCN 1.5 h; or (2) Os 24 h → CaC 24 h → FeCN 1.5 h. After staining, the samples were dehydrated and embedded in Sparre's resin as described above for the 2 mm samples. After the resin hardened, they were imaged with μCT, then trimmed flat to expose the top surface, smoothed, and imaged with low-vacuum SEM and EDS. For EDS measurements, we chose a point measurement in the neuropil in the center of the sample.

[0194] Interaction of Pg incubation with Os-FeCN gradient (H3) Brain hemisphere samples (H3) were stained according to the following steps: 1st Os 4°C 96 h, room temperature 24 h → CaC 4°C 48 h → FeCN 4°C 48 h → 2nd Os 72 h → CaC 24 h → H2O 29 h → Pg 24 h → H2O 48 h → 3rd Os 48 h → 4%UA 4°C 48 h, 50°C 5 h → H2O 42 h. For all CaC and H2O steps, the corresponding solutions were changed every 4 hours / overnight (i.e., once in the morning, midday, and afternoon). During staining, brain hemispheres were imaged by μCT at the main staining steps (after FeCN, after 2nd Os, and after Pg). After staining, brain hemispheres were cut into approximately 2 mm coronal sections with a razor blade. The coronal sections were then dehydrated and embedded in Spurr's resin as described above for the 2 mm samples. After the resin cured, they were trimmed flat to expose the top surface, smoothed, and imaged in a high vacuum SEM.

[0195] Pg incubation and its interaction with Os-FeCN gradient and CaC (H1,2) Two brain hemisphere samples (H1,2) were stained according to the following steps: 1st Os 4°C 63 h, room temperature 24 h → CaC 4°C 24 h → FeCN 4°C 48 h → CaC 24 h → 2nd Os 32 h → CaC 17 h → H2O 9 h → Pg 48 h → H2O 40 h → 3rd Os 48 h → 4%UA 4°C 48 h, 50°C 5 h → H2O 48 h. In all CaC and H2O steps, the corresponding solutions were changed every 4 h / overnight (i.e., once in the morning, midday, and afternoon). During staining, the brain hemispheres were imaged by μCT at the main staining steps (after FeCN, after 2nd Os, and after Pg). After staining, the brain hemispheres were cut into approximately 2 mm coronal sections with a razor blade. The coronal sections were then dehydrated and embedded in Spurr's resin as described above for the 2 mm specimens. After the resin hardened, they were trimmed flat to expose the top surface, smoothed, and imaged in a high-vacuum SEM.

[0196] Effect of 2 days of CaC incubation at room temperature on the Os-FeCN gradient (H4, 5, 6) Three brain hemisphere samples (H4, 5, 6) were stained according to the following steps: 1st Os 4°C 72 h, room temperature 24 h → CaC 48 h → FeCN 4°C 48 h → CaC 24 h → 2nd Os 48 h → CaC 24 h → H2O 24 h → Pg 24 h → H2O 24 h → 3rd Os 24 h → 4%UA 4°C 48 h, 50°C 5 h → H2O 48 h. In all CaC and H2O steps, the corresponding solutions were changed every 4 hours / overnight (i.e., once in the morning, midday, and afternoon). During staining, brain hemispheres were imaged by μCT at the main staining steps (after FeCN, after 2nd Os, and after Pg). After staining, H5 was cut into approximately 2 mm coronal sections with a razor blade. The coronal sections were then dehydrated and embedded in Spurr resin as described above for the 2 mm specimens. H4, 6 were embedded in Spurr resin. After the resin cured, H5 was trimmed flat to expose the top surface, smoothed, and imaged in a high vacuum SEM. H4, 6 were trimmed flat to expose the center, smoothed, and imaged in a high vacuum SEM.

[0197] Effect of incubation of CaC at 4℃ for 4 days on the Os-FeCN gradient (H13) Brain hemisphere samples (H13) were stained according to the following steps: 1st Os 4°C 68h → CaC 4°C 96h → FeCN 4°C 72h → CaC 48h → 2nd Os 48h → CaC 24h → H2O 24h → Pg 24h → H2O 24h → 3rd Os 41h → 4%UA 4°C 48h, 50°C 12h → H2O 24h. In all CaC and H2O steps, the corresponding solutions were changed every 4h / overnight (i.e., once in the morning, midday, and afternoon). During staining, brain hemispheres were imaged by μCT at the main staining steps (after 2nd Os, after Pg). After staining, brain hemispheres were embedded according to Spar's resin embedding method for brain hemispheres. After resin hardening, it was trimmed to expose the center, the surface was smoothed, and imaged by high vacuum SEM.

[0198] Whole brain staining (W1) Mouse whole brain samples (W1) were stained in the following steps: 1st Os 4°C 96h → CaC 4°C 168h → FeCN 4°C 72h → CaC 4°C 48h, room temperature 48h → 2nd Os 48h → CaC 72h → H2O 48h → Pg 24h → H2O 48h → 3rd Os 96h → 4%UA 4°C 48h, 50°C 5.5h → H2O 24h. In all CaC and H2O steps, the corresponding solutions were changed every 4h / overnight (i.e., once in the morning, midday, and afternoon). During staining, the brains were imaged by μCT at the main staining steps (during 1st Os, after 2nd Os, and after Pg). After staining, the brains were cut into coronal sections of approximately 2mm thickness. The sections were then embedded according to Spurr's resin embedding method for 2mm specimens. After the resin hardened, the sections were trimmed flat to expose the surface, smoothed, and examined under high vacuum SEM.

[0199] Effect of the second Os step on sample stability in HO Two groups of 2 mm samples (these groups differ in whether they were exposed to the 2Os step) were incubated under the following conditions: (1) 1Os 24 h → CaC 24 h → FeCN 24 h → 2Os 3 h → CaC 0.5 h → HO (0.5 h twice) → HO; or (2) 1Os 24 h → CaC 24 h → FeCN 24 h → CaC 0.5 h → HO (0.5 h twice) → HO. Samples were then stored in HO and imaged by optical microscopy at various time points (from 2 h to 100 h) to examine the macroscopic integrity of the samples.

[0200] Pg-Os(vi) interaction Mouse brain hemisphere samples were stained according to one of the following steps: (1) Os 4°C 72h → CaC 4°C 96h → FeCN 4°C 72h → CaC 48 → Os 48 → CaC 24 → CaC 24 → Pg 24 (see Table 4, experiment 28, Fig. 10a, b); (2) Os 4°C 72 → CaC 4°C 96 → FeCN 4°C 72 → CaC 48 → CaC 48 → CaC 24 → CaC 24 → Pg 24 (see Table 4, experiment 29, Fig. 10a, c); (3) Os 4°C 72 → CaC 4°C 96 → FeCN 4°C 72 → CaC 48 → Os 48 → KCl 24 → KCl 24 → Pg 24 (see Table 4, experiment 30, Fig. 10a, d); (4) Os 4°C 72 → CaC 4°C 96 → FeCN 4°C 72 → CaC 48 → Os 48 → KCl 24 → KCl 24 → Pg 24 (see Table 4, experiment 30, Fig. 10a, d). → CaC 4°C 96 → FeCN 4°C 72 → CaC 48 → Os 48 → KCl 24, pH 7 → KCl 24, pH 7 → KCl pH1 → KCl 24, pH 7 → Pg 24 (see Table 4, experiment 31, Fig. 10a, e). After Pg, the samples were scanned with μCT using the parameters described in the μCT method section below.

[0201] Brain hemisphere samples from condition 2, i.e., "(2)" above, were then cut into approximately 2 mm coronal blocks for further processing. The coronal sections were rinsed in CaC for 24 hours and then stained with Os in HO (see Table 4, experiment 32, and Figure 10f) or Os in CaC (see Table 4, experiment 33, and Figure 10g) for 24 hours. They were then embedded following the 2 mm Spurr resin embedding protocol. After the resin cured, the samples were cut in half and checked for membrane contrast with low vacuum SEM.

[0202] Dehydration of 2mm sample After the final water rinse step of the staining protocol, the 2 mm samples were exposed to a graded dehydration series in ethanol (50% at 4°C, 75% at 4°C, and 100% at room temperature; each step was 45 min) and then incubated three times in acetone (45 min each).

[0203] Dehydration of the brain hemispheres After the final water rinsing step of the staining protocol, brain hemisphere samples were exposed to a graded dehydration series in ethanol (25% 4°C, 50% 4°C, 75% 4°C, 100% room temperature; each step 8 h / overnight) and then incubated three times in acetone (each 8 h / overnight).

[0204] Dehydration of whole brain samples After the final water rinsing step of the staining protocol, whole brain samples for subsequent whole-volume embedding (e.g., W3) were exposed to a graded dehydration series in ethanol (25%, 12 h at 4°C, 50%, 75%, 100%; each step for 24 h at 4°C) and then incubated three times in acetone (24 h each).

[0205] Infiltration of Sparre's resin and embedding of 2 mm specimens After dehydration, Spurr resin (0.95g ERL 4221, 5.9g DER 736, 0.1g NSA, 113ul DMAE) was incubated stepwise in 25%, 50% and 75% acetone (8 hours or overnight for each step). The samples were then incubated 4 times in 100% Spurr resin (8 hours or overnight for each step). Finally, they were embedded in freshly prepared Spurr resin and cured at 70°C for 1-3 days. All steps were performed at 4°C. For each resin change, the tubes were removed from the refrigerator and allowed to warm to room temperature for 20-30 minutes.

[0206] Spurr's resin infiltration and embedding of brain hemisphere samples After dehydration, graded incubations of Spurr resin (0.95g ERL 4221, 5.9g DER 736, 0.1g NSA, 113ul DMAE) were applied in concentrations of 25%, 50% and 75% in acetone (24h for each step). The samples were then incubated for 2 days in 90% resin, 3 days in 95% resin, 3 days in 100% resin, and then incubated 4 times in 100% Spurr resin (8h or overnight each time). Finally, they were embedded in freshly prepared Spurr resin and cured at 70°C for 1-3 days. All resin steps were performed at 4°C. For each resin exchange, the tubes were removed from the refrigerator and allowed to warm to room temperature for 20-30 min.

[0207] Spurr's resin infiltration and whole-volume embedding of whole brain samples After dehydration, graded incubations of Spurr resin (0.95g ERL 4221, 5.9g DER 736, 0.1g NSA, 113ul DMAE) in acetone at concentrations of 25%, 50% and 75% were applied (25% and 50% for 2 days, and 75% for 3 days). Next, whole brain samples (e.g. W3) were incubated in 90% resin for 2 days, 95% resin for 4 days, and 100% resin for 6 days. The 95% and 100% resin solutions were changed every 2 days. Finally, they were embedded in freshly prepared Spurr resin and cured at 70°C for 3 days. All resin steps were performed at 4°C. For each resin change, the tubes were warmed to room temperature 20-30 min after removal from the refrigerator.

[0208] Measuring the viscosity of Epon resin 10 ml of Epon resin (Sigma-Aldrich, ratio: 5.9 g Epon medium, 2.25 g DDSA, 3.7 g MNA, 205 ul DMP) was prepared and stored at 4° C. or room temperature in 15 ml Falcon tubes. At various time points, the resin was videotaped moving within the tube after inverting the tube. The distance traveled by the resin within the tube was measured using the graduations (in ml) printed on the Falcon tube. The rate of resin movement was used as a measure of the viscosity of the resin.

[0209] Infiltration with Epon resin and embedding of 2 mm specimens After dehydration, the samples were incubated in pure acetone three times (45 min each). Then, Epon resin (5.9 g Epon medium, 2.25 g DDSA, 3.7 g MNA, 205 ul DMP) was incubated in graded concentrations of 12.5%, 25%, 37.5%, 50%, 62.5%, 75%-87.5% in acetone (4 h or overnight for each step). Next, the samples were incubated 4-6 times in 95% Epon resin (8 h or overnight for each step), then 4 times in 100% Epon resin (8 h or overnight for each step), before embedding and curing at 60°C for 1-3 days. All steps of the resin were performed at 4°C. For each resin exchange, the tubes were warmed to room temperature 20-30 min after removal from the refrigerator.

[0210] Optical microscopy imaging of sample surfaces to evaluate resin gradients Resin-embedded samples (2–3 mm / brain hemisphere) were trimmed to expose the center, and the surface was smoothed with a diamond knife. Optical microscope images of the sample surface were taken with the image plane tilted slightly so that the originally black sample surface appeared silvery.

[0211] μCT volumetric imaging of samples without resin embedding Instead of performing resin infiltration, 2 mm samples were embedded in 2% agarose (Sigma-Aldrich) in 0.15 M CaC or water (depending on the last staining step the sample was exposed to) in 2 ml Eppendorf tubes to allow imaging with μCT without the need for resin embedding. These were imaged in μCT (Zeiss Xradia 520 Versa) using a voltage of 80 kV with a voxel size of 3–6 μm. For μCT imaging of brain hemispheres, the samples were placed in a 50 ml glass tube. The glass tube was then placed in a 140 ml syringe to keep it stable during μCT imaging (using a voxel size of 10–60 μm).

[0212] Low-vacuum SEM imaging of incompletely stained samples To study samples at intermediate staining steps where staining was not complete (and therefore signal and conductivity often decreased), they were embedded in resin. They were then trimmed with a diamond head trimmer (Leica EM TRIM2) to expose the center at the block face of the sample; the block face was smoothed with a diamond knife ultramicrotome (Leica EM UC7); and then imaged with a scanning electron microscope (Quanta FEG 450, FEI Company) equipped with a field emission cathode and low vacuum mode. The chamber pressure was set at 30 Pa. For the incident electron beam, a spot size of 3.5 and an acceleration energy of 5 keV were used, with a pixel dwell time of 8–20 μs and an in-plane pixel size of 5.62 nm. 2 (Corresponding electron dose: 112~280e without considering electron loss due to the skirt effect of low vacuum) - / nm 2 ) or in-plane pixel size 11.24 nm 2 (Corresponding electron dose: 28~70e without considering electron loss due to the skirt effect of low vacuum) - / nm 2 ) and imaging was performed using a backscattered electron CBS detector at a working distance of approximately 5 mm.

[0213] High vacuum SEM imaging of fully stained samples For fully stained and resin-embedded specimens (and therefore expected to exhibit sufficient conductivity), trimming and smoothing were similar to the previous section, but SEM imaging was performed in high vacuum mode (5 × 10 -4 The electron beam was injected at a 3.5 Pa with a spot size of 3.5 and an acceleration energy of 2.8 keV, with a pixel dwell time of 6–8 μs and an in-plane pixel size of 11.24 nm. 2 (Corresponding electron dose: 16~21e - / nm 2 ) or 5.62 nm 2 (Corresponding electron dose: 64~84e - / nm 2 ) and imaging was performed using a backscattered electron CBS detector at a working distance of approximately 5 mm.

[0214] EDS analysis Resin-embedded samples were trimmed to expose the center as a block face (Leica EM TRIM2), smoothed (Leica EM UC7), and coated with a 10 nm gold layer (Leica EM ACE600); they were then imaged with a scanning electron microscope (Amray 1830) equipped with a Si(Li)EDS detector. An incident electron beam with an energy of 18 keV was used with a working distance of 15-20 mm and a take-off angle of 20.4 degrees. Spectral collection times ranged from 20 to 90 s.

[0215] Acquisition of ultraviolet-visible (UV-vis) spectra of Os(vi) solutions 1% potassium osmate(vi) in 0.15 M sodium cacodylate buffer was prepared by adding 0.05 g potassium osmate(vi) powder (Sigma-Aldrich) to 5 ml cacodylate buffer. The solution was diluted 100-fold and placed in a glass cuvette to avoid clipping of the spectral signal. Measurements were performed on a UV-Vis spectrometer (Jasco V-670) using a wavelength range of 190–11,400 nm. Measurements on the same solution were performed at 2 min, 2 h, and 24 h.

[0216] Raman spectrum measurement of Osvi+FeCN solution The inventors have determined that the wavelength range is 0 to 4,400 cm -1 The following chemicals were measured undiluted on a custom-built Raman spectrometer from Goethe University Frankfurt (Faculty of Chemistry): (1) 0.15 M sodium cacodylate buffer; (2) 1% potassium osmate(vi) in 0.15 M cacodylate buffer; (3) 0.3% potassium osmate(vi) and 1.3% potassium ferrocyanide(ii) (Sigma) in 0.15 M cacodylate buffer. Aldrich); (4) 2.5% potassium ferrocyanide(ii) in 0.15 M cacodylate buffer; (5) 1.9% potassium ferricyanide(iii) in 0.15 M cacodylate buffer (Sigma-Aldrich); (6) Staining solution of 2 mm specimens in 2 ml Eppendorf tubes, 2% OsO4 (Serva) in 0.15 M cacodylate buffer for 24 h, followed by washing with 0.15 M cacodylate buffer for 1 h, and 2.5% potassium ferrocyanide(ii) in 0.15 M cacodylate buffer for 17 h.

[0217] Quantification of membrane contrast Membrane contrast was determined as the difference in pixel intensity between membrane and non-membrane regions in EM images. For this, 500 × 500 pixel regions were taken from raw EM images (Fig. 1c–e) and brightness normalized (1–99 percentile, Matlab, imadjust). One randomly selected 500 × 500 subimage was then used to train a random forest classifier (Fiji, trainable Weka segmentation (NPL39)) to automatically identify membrane and non-membrane voxels. This classifier was then applied to all subimages and membrane contrast was calculated as the difference in median intensity between membrane and non-membrane voxels. To determine differences between measurement conditions, a two-way ANOVA was performed in GraphPad Prism and Tukey's multiple comparisons were calculated between staining protocols and sampling locations. Next, to determine the significance of the observed differences, we performed one-tailed t-tests comparing membrane contrast between the Hua protocol (NPL11) and the new protocol (pooled Os3 and Os24) at the core and intermediate positions where improvement was predicted.

[0218] 3D EM imaging and image alignment Brain hemispheres H6 and H13, approximately (1.5 mm) 3 Partial volumes of size 100 nm were cut with a Leica EMTrim-2. SBEM datasets were acquired using a custom-built SBEM microtome (NPL5) mounted inside the chamber of a scanning electron microscope (FEI Quanta, Thermo Fisher Scientific, USA). Image acquisition and the SBEM microtome were controlled using custom-built software (NPL41). Aperture 4 (spot size 3.5, beam current 160 pA, dwell time 2.8 μs, electron dose 22 e - / nm 2 ) or aperture 6 (spot size 3.0, beam current 47 pA, dwell time 12 μs, electron dose 28 e - / nm 2 ) using an incident electron beam with an acceleration energy of 2.8 KeV (11.24 nm) 2 Imaging was performed with an in-plane pixel size of

[0219] Image alignment of all EM datasets and their segments was performed using routines described in (NPL40). Axon tracing was performed with webKnossos (NPL42). Briefly, axonal tracings were performed using (3 μm) sections from the central H6 striatal dataset or the control striatal dataset. 3 bboxes were randomly inoculated with 20 axons. Tracing was performed manually by three experienced annotators. The occurrence of discontinuities (defined as failure to trace a non-terminal node) was recorded. The mean inter-error distance was calculated as the total path length (mm) divided by the number of discontinuous nodes. Permutational random resampling was performed to calculate the 25th and 75th percentiles for each condition.

[0220] ATUM study of fully embedded whole brain samples (W3) Mouse whole brain samples (W3) were stained according to the whole brain staining protocol described herein (see Tables 1 and 2). After staining, the whole brain samples (W3) were dehydrated and embedded as previously described (see sections "Dehydration of Whole Brain Samples" and "Spurr's Resin Infiltration and Total Embedding of Whole Brain Samples" above). The samples were then embedded in freshly prepared Spurr's resin and cured at 70°C for 3 days.

[0221] To examine the resin quality in the center of the sample, after the resin hardened, the whole brain sample was cut into two equal halves. The exposed sample surface was then trimmed into a hexagon of approximately 4 × 2 mm, and then ultrathin sections with a section thickness of 38 nm were cut in ATUM. 290 consecutive cuts (>10 μm series) were performed.

[0222] Example 2 - Results Here, we report protocols for reliable en bloc staining of 2-3 mm sized specimens (Figure 1), mouse brain hemispheres (Figure 2), whole mouse brains (Figures 3 and 14), and human cortical samples (Figure 11). Their development required simultaneously solving the following problems: recurrent staining inhomogeneity, sample instability leading to breakage (especially for brain hemisphere and whole brain specimens), and uniform resin infiltration, which are explained below.

[0223] For the development of the protocol, we first used X-ray microtomography (μCT) images to evaluate the staining gradient (NPL19, NPL31). In addition, low-vacuum SEM (NPL14, NPL30) was applied to check the ultrastructural contrast after an intermediate step in the staining experiment, the stage where the sample is charged in a high-vacuum SEM. This was particularly important because samples that showed a homogenous appearance in μCT may reveal poor membrane contrast or damaged ultrastructure when analyzed by EM (Supplementary Figures 5, 8).

[0224] First, we applied the available 1 mm protocol (NPL11) to a 2 mm sized specimen (Fig. 1b), which caused a strong staining gradient (Fig. 1c, Fig. 4a, b) and incomplete resin penetration (Fig. 1f). We then used μCT to investigate at which step of the protocol the gradient occurred and found two layers of gradient after OsO4-FeCN incubation and an additional third gradient layer after TCH-OsO4 incubation (Fig. 4c). An obvious remedy in observing such gradients was to extend the duration of the respective incubation steps. Extending the FeCN step from 1.5 h to 12-17 h indeed eliminated the gradient (Fig. 4d).

[0225] Surprisingly, however, omitting the FeCN step entirely and simply incubating with OsO4 for 3 h did not decrease the membrane contrast (and there was no gradient) under SEM (Fig. 5a). Only when the OsO4 incubation was also extended to 24 h did the FeCN step enhance the membrane contrast (Fig. 5b; see Example 3 and Fig. 9d, e for a possible explanation of this phenomenon in terms of Os(vi)-CN coordination chemistry).

[0226] Although the FeCN-induced gradient could be eliminated in this manner, the TCH-induced gradient could not be similarly resolved; rather, extended TCH incubation caused the samples to break down, most likely due to gaseous products generated by the reaction of OsO4 with TCH, as previously reported (NPL19) (Figure 4e).

[0227] We therefore adopted the replacement of TCH with pyrogallol (Pg), as proposed in the prior art (NPL19). When replacing TCH with Pg in the 1 mm protocol (NPL11), sufficient sample conductivity and contrast were found (Fig. 4f, g, h). It was confirmed that the pyrogallol incubation must be performed in HO and not in cacodylate buffer (CaC), otherwise the membrane contrast is reduced (NPL19) and a staining gradient is created (Fig. 4i). Furthermore, it was found that the interaction of CaC with pyrogallol can result in gradients and breakage, so that the CaC needs to be pre-incubated in HO before the pyrogallol step to be washed out (Fig. 6d). When the incubation time of pyrogallol and the subsequent OsO4 step was extended, the second gradient disappeared (Table 1, Fig. 4j).

[0228] Regarding the final steps of this protocol, uranyl acetate (UA) and lead aspartate (Ld), we confirmed that a slight gradient was induced by UA under μCT (Fig. 4k). This gradient was eliminated by increasing the UA concentration from 2% to 4% (Fig. 4k). Because the low concentration of Ld resulted in insufficient diffusivity (20 mM / L lead nitrate dissolved in 30 mM / L saturated aspartate buffer at a 2:3 molar ratio (NPL34)), the final lead aspartate step was omitted (Fig. 4k). Moreover, even when the lead aspartate step was omitted, the conductivity and staining contrast of the sample were sufficient (Fig. 1d, e). This allowed us to obtain a high-contrast, uniform staining protocol for samples with a size of 2 to 3 mm (Fig. 1e, Table 1; contrast quantification in Fig. 1h).

[0229] Next, it was necessary to ensure uniform resin infiltration. We used Spar resin (NPL5) for samples to be cut and imaged using a serial block-face scanning electron microscope (SBEM) and looked at an Epon 812 substitute (hereafter referred to as Epon) for samples to be cut using an automated tape-collecting ultramicrotome (ATUM) (NPL8). We took into account the fact that the epoxy resin blender could polymerize during infiltration. This would increase the viscosity of the blender, and once the polymerization process had passed the gel point, further diffusion would not be possible (NPL33). Therefore, a practical measure to improve resin infiltration was to slow down the polymerization reaction and keep the viscosity low. To do this, all resin infiltration steps were kept at 4 °C (NPL33), which indeed slowed down the polymerization process significantly (Figure 1g). Furthermore, we added a step of placing 95% resin in 5% acetone before incubating the sample in pure resin; this small amount of acetone was shown to significantly reduce the viscosity of the resin (NPL18). Finally, the incubation time of the resin (Epon) was extended from 1 day to 4-5 days (Figure 1g; Table 2, which also contains data for Spurr resin). With these modifications, we were able to successfully infiltrate both Spurr and Epon epoxy resin into the center, resulting in a uniformly embedded 2 mm-sized specimen (Figure 1f). [Table 1]

[0230] Next, the inventors applied the resulting protocol to a 25- to 60-fold larger volume (approximately 5 × 10 × (5 or 10) mm 3 We hoped to apply this technique to staining mouse brain hemispheres and whole brain samples (Figs. 2a, 3a). In developing the protocol, we used mouse brain hemispheres as a surrogate target (Fig. 2), since whole mouse brains are symmetrical replicas of the hemispheres, given their nominally similar access geometry, and later tested the results of an exemplary mouse whole brain staining (Fig. 3).

[0231] For mouse brain hemispheres, we first extended the initial OsO4 incubation to 3 and 6 days, since we had already noticed a strong contrast enhancement with prolonged incubation with OsO4 in 2-3 mm sized samples (see above, Fig. 1d, e, Fig. 5a, b). Indeed, 3 or 6 days of OsO4 incubation increased membrane contrast even in the center of the sample. However, we encountered a significant obstacle: when OsO4 was incubated long enough to reach the center of the brain hemisphere sample, the outer parts of the sample showed strong signs of ultrastructural collapse (Fig. 2b, note: these were not evident in μCT, only in EM images). Most of the neurites and the cytoplasm of the cell bodies appeared to have been extracted, probably indicating that intracellular proteins had been removed (Fig. 2b, note that NPL7 has already considered this for overfixation of tissue with OsO4). Because the outer parts of the sample correspond to the cortical grey matter, which is the key target for whole-brain connectomics analysis, whole-brain staining with good stain penetration but poor ultrastructural quality in the cortical periphery would be useless.

[0232] To overcome this obstacle, we adopted the view that the ultrastructural changes were probably the result of protein overoxidation due to prolonged incubation with OsO4 (see Supplementary Material) and that this could be delayed by lowering the incubation temperature (4 °C) (NPL7). Indeed, samples incubated at 4 °C showed no obvious OsO4-based cytoplasmic changes even after 7 days of incubation in OsO4 (Fig. 5e, f). A sufficiently long OsO4 incubation at 4 °C (step length: diffusion time + additional incubation time of about 1 additional day of brain hemisphere) made a step to room temperature unnecessary, and yet subsequent FeCN gave enhanced membrane contrast (Fig. 5g, h, i, j; see Supplementary Material Fig. 9d, e for a possible chemical explanation). FeCN incubation itself also caused significant tissue damage at room temperature (Fig. 5g). We therefore also incubated FeCN at 4 °C, solving this problem (Fig. 5j).

[0233] Regarding the FeCN step, we noticed an additional problem: especially for large specimens such as brain hemispheres, we observed an inverse gradient of low μCT image intensity in the periphery (Fig. 6b) and a very slow diffusion of FeCN (Fig. 6d). This could be the result of a direct redox reaction between OsO4 and FeCN (see Example 3). However, when we added a washing step with CaC between OsO4 and FeCN incubation to avoid direct interaction, the resulting gradient was removed, the staining intensity in the periphery remained high (Fig. 6c) and the diffusion of FeCN was much faster (Fig. 6e, f). We noticed that these washing steps had to be performed for a sufficient time at 4 °C (Fig. 6h); if these steps were not long enough, a staining gradient remained, which could be amplified in the later pyrogallol step and contribute to the breakage (Table 1, Fig. 6g, h). If this was done at room temperature, the incubation time would be shorter (Figure 6h), but the samples were less stable in water, the cerebellar tissue was damaged (Figures 13f-g), and we also observed a decrease in PSD staining (Figure 9). A washing step at room temperature also seemed to decrease PSD staining.

[0234] After addressing the issue of OsO4 penetration, we were left with one major obstacle: large samples consistently break during the staining process, either breaking into several small fragments altogether, or developing micro-fractures that prevent the reconstruction of dense circuits (Fig. 2c, d). These breaks usually occur in association with the pyrogallol incubation step (Table 1). It was therefore necessary to evaluate the impact of all previous protocol steps on the stability of the samples during pyrogallol incubation. This was particularly important because, as confirmed for the 2 mm samples, the pyrogallol incubation (including pre- and post-wash steps) must be performed in HO, and this HO incubation can generate significant osmotic pressure in the samples. We found that inserting an additional prolonged incubation step of OsO4 after FeCN stabilized the samples, so that they could be incubated in HO for up to 100 h without significant breakage occurring (Fig. 6i, a possible explanation is membrane perforation induced by OsO4, which increases their resistance to osmotic stress, NPL7). This additional OsO4 step also serves to enhance background staining and thus increase sample conductivity in the SEM, although the most important aspect is the improved stability of large samples (see Example 3, section "Additional modified protocol for very fragile samples" together with Figure 10 for an alternative approach that omits the second osmium step and avoids the pure HO incubation).

[0235] We applied the brain hemisphere protocol to n=3 brain hemispheres (Fig. 2, Fig. 7, Fig. 12). All of these remained intact, and uniform high-contrast staining was obtained throughout the sample. Furthermore, whole mouse brains with slightly extended incubation times were stained (Table 1, Fig. 3), thus obtaining a whole mouse brain staining protocol for large-scale connectomics (NPL1). We observed two types of remaining artifacts in the staining of large-scale samples (Fig. 13): the detachment of large vessel walls from the surrounding neuropil and the slight remaining micro-breakage in subcortical regions with a high proportion of myelinated fibers. Also, special care had to be taken to maintain the integrity of the cerebellum (Fig. 13f, g). None of these remaining issues are expected to substantially affect the connectomics reconstructions.

[0236] We performed a series of automated ultramicrotome sectioning (as described in NPL8) tests on the central section of a fully stained and fully embedded whole mouse brain sample (W3) and confirmed that the whole-volume embedding protocol described herein also affords sufficient sample stability for ultrathin sectioning (Figure 14c, d, e).

[0237] To quantitatively determine the sufficiency of staining quality and resin stability in the sample core for 3D EM acquisition and axonal reconstruction, we acquired an SBEM dataset from the core of one stained, whole-volume embedded mouse brain hemisphere (striatum) ( Fig. S12 , H6) and replicated it in a 1 × 1 × 0.5 mm 3D EM specimen stained with the Hua protocol (NPL11). 3 We compared this to a control SBEM dataset of the striatum obtained from a 35-mm sample. Both manual (Fig. S12d) and automated (Fig. S12e) axonal reconstructions yielded similar results between the locally stained control and the brain hemisphere stained samples.

[0238] Finally, we applied our protocol to a human brain biopsy sample to directly test the applicability of the protocol to large human brain samples (Figure 11, tissue obtained at (NPL40), patient H6). We performed a 2x3x2mm section of human frontal cortex spanning all cortical layers. 3 (i.e. 12mm 3 We stained and embedded a human specimen of size 10 mm (3.5 mm) in size. The tissue was stained uniformly throughout the layers (Fig. 11c). Furthermore, we used ATUM to acquire a section series (2733 sections with a cut thickness of 35-40 nm from the sample center) testing the sectionability of large-scale 3D EM.

[0239] For this 2x3x2mm size of human samples, some incubation steps are longer compared to the 2x2x2mm protocol (see Table 2). In general, it is advisable to interpolate incubation times between the reported 2mm protocol and the brain hemisphere protocol for samples with dimensions greater than 2mm or irregular shapes. For ATCM sections, it is also advisable to increase the resin infiltration time compared to SBEM.

[0240] We believe that the protocols reported here will be useful for large-scale connectomics projects in mice and other species. In particular, mouse brain volumes approach the relevant cortical and subcortical volumes of higher mammals such as non-human primates and even humans. The ultimate goal of obtaining large connectomes from parts or the entire human cortex (NPL21) will also benefit from the advances described here, which will be useful in both basic research and clinical applications, and also for routine connectomics analysis of pathological brain specimens, e.g., following neurosurgical interventions (NPL40).

[0241] Example 3 - Further Results Chemical concepts of OsO4-FeCN-related membrane contrast enhancement Since the 1970s, potassium hexacyanoferrate(II) (FeCN) has been used in combination with OsO4 for membrane contrast enhancement of biological specimens in electron microscopy (NPL3, NPL13). Chemically, this was thought to involve a redox reaction between OsO4 and FeCN (NPL3, NPL13, NPL28, NPL36), and so this protocol has been called the "reduced osmium" (rO) protocol. Two mechanisms were considered for contrast enhancement in the rO protocol: either more osmium compounds were deposited in the membrane (NPL3, NPL4, NPL36, NPL37) or cytoplasmic background osmium compounds were removed (NPL17, NPL22, NPL37). More recent improved staining protocols were based on the following chemical concept (NPL11): the staining process was considered to be a two-step chemical reaction: first, FeCN was converted to Osv iii O4 can be converted to its lower valence form, Os vi O2(OH)4 2- (Os(vi)). Then, two molecules of Os(vi) are reduced to one molecule of OsO4 and one molecule of Os IV It was thought that OsO2 could be dismutated to O2 and deposited on membranes (NPL11), resulting in enhanced membrane contrast.

[0242] For the protocol reported here, we developed a different chemical concept. First, it was pointed out that there are several experimental results that do not support the explanation of "redox disproportionation" (NPL11) mentioned above. The postulated disproportionation reaction was confirmed either in organic solvents or in low pH aqueous solutions (NPL15), but this is less likely to occur in cacodylate buffer (CaC). In support of this, we show that potassium osmate(vi) (K2[Os VIWe found that the UV-visible absorption spectrum of the OsO4-FeCN [O2(OH)4] solution was unchanged after 24 h compared to freshly prepared solutions (Fig. 8d). In brain tissue, disproportionation may still occur in a more complex manner involving interactions with lipid membranes. However, we found that direct staining of the specimens with potassium osmate(vi) in CaC solution did not enhance membrane contrast (Fig. 8c). Furthermore, we noticed that the redox reactions between OsO4 and FeCN (NPL3, NPL11, NPL13, NPL28, NPL36) underlying the membrane contrast enhancement have little direct support. A direct test would be to determine membrane contrast by suppressing the redox reaction. We attempted to suppress the OsO4-FeCN redox reaction by adding a CaC washing step to remove OsO4 from the tissue before adding FeCN, and observed a similar membrane contrast (compare Fig. 8a,b with Fig. 1d,e). This indicates that the OsO4-FeCN redox reaction may not be a prerequisite for the enhancement of film contrast.

[0243] To develop an alternative explanation for membrane contrast enhancement, we were guided by the observation that the duration of the OsO4 incubation step was critical for the effect of FeCN: if the OsO4 incubation time was short (but sufficient to uniformly stain a 2 mm-sized sample, e.g., 3 h), adding FeCN did not actually enhance membrane contrast (Fig. 5a); only when the OsO4 incubation was extended to 24 h was the following FeCN step effective in membrane contrast enhancement (Fig. 5b). What processes could have occurred during the extended OsO4 incubation? After a short incubation, both the membrane and the background appeared to be stained (Fig. 5a), indicating that the osmium compound was deposited / attached to both targets. However, after prolonged incubation, the cytoplasmic background (presumably composed mainly of proteins) may have been altered by OsO4 to produce products that FeCN could react with and wash away, resulting in reduced background staining (Fig. 5b; consistent with previous studies (NPL17, NPL22, NPL37)). One possible process / reaction between OsO4 and the background is overoxidation of proteins (NPL7), which may induce 3D structural changes in proteins (e.g., degelation (NPL27)). As a result, more protein-bound osmium (valence vi (NPL7)) is exposed to the aqueous phase, allowing interaction with FeCN in aqueous solution. This notion that structural instability of proteins is a prerequisite for the FeCN effect is supported by experiments with the addition of CaCl2 as a protein-crosslinking cation (NPL7), which was indeed found to inhibit the membrane contrast enhancement effect of FeCN after prolonged OsO4 incubation (Fig. 8g). Moreover, incubation at 4 °C is expected to make the protein structure more stable and the chemical reactions slower. Indeed, low-temperature incubation could inhibit the effect of FeCN on the membrane contrast after long-term OsO4 incubation (Fig. 8h).

[0244] However, is the addition of FeCN after prolonged OsO4 exposure essential for membrane contrast enhancement, or would a long OsO4 incubation alone provide sufficient membrane contrast? Samples were stained with even longer OsO4 incubations (from 24 h to 3–6 days) and then examined for membrane contrast by low-vacuum SEM. After 6 days of OsO4 incubation, membrane contrast enhancement was indeed realized (Fig. 5c, d). However, extensive background extraction also occurred (Fig. 5c, d). Taken together, we can interpret this as an indication that FeCN is not strictly necessary to enhance membrane contrast, but may accelerate contrast enhancement so that sufficient contrast can be obtained before background is extracted by OsO4 incubation ("overfixation").

[0245] Nevertheless, we had to hypothesize about the actual reaction between osmium compounds and FeCN. These experiments and concepts are described below.

[0246] During prolonged OsO4 incubation, a pink reaction product was observed diffusing out of the sample (OsO4 was yellowish in solution by comparison) (Figure 9a), which is the typical color of osmate(vi) in cacodylate. This further supports the previous idea that the background osmium species exposed by prolonged OsO4 is Os(vi). During very long OsO4 incubation, as the protein becomes further oxidized, exposed Os(vi) may have been released into the aqueous phase and stabilized Os(vi) in water by coordinating with anions in the CaC buffer (Figure 8d). In this coordinated form, Os(vi) can diffuse out of the sample to reduce background staining and enhance membrane contrast. This means that this Os(vi) removal process is slow and does not enhance the membrane contrast after 24 h of OsO4 incubation, but only after 3–6 days of OsO4 incubation (Fig. 5b), which is consistent with the idea from previous studies that the diffusion of “reduced osmium” is slow (NPL11, NPL19).

[0247] How does the addition of FeCN accelerate this Os(vi) background removal process? After OsO4 incubation, we expected Os(viii) and Os(vi) to move freely in the aqueous phase. Since redox reactions or disproportionation, the key processes in the above explanation, seemed unlikely (see above), the possibility of reactions between Os(viii)-FeCN or Os(vi)-Os(vi) was ruled out; we had to consider that a remaining possible chemical reaction must occur between Os(vi) and FeCN. This is consistent with the observation that the staining solution after the FeCN step was a blue-green color similar to the color observed in the in vitro reaction of Os(vi) with FeCN (Fig. 9a).

[0248] What is the actual reaction occurring between Os(vi) and FeCN? This was investigated by measuring the Raman spectrum of a mixed solution of Os(vi) and FeCN. In the mixture, the expected spectral peaks for potassium hexacyanoferrate(iii) (FeCNiii) were not observed (Figure 9b), indicating that Os(vi) was not able to oxidize FeCN to FeCNiii. On the other hand, Os(vi) was able to oxidize FeCN to FeCNiii by OH - (NPL11, NPL25) and CN - (NPL6, NPL23, NPL24, NPL25), and FeCN is unstable and spontaneously dissociates slowly in aqueous solution to give free CN. - (NPL32). Thus, a possible reaction between Os(vi) and FeCN is a coordination reaction, where Os(vi) is converted to CN from FeCN. - competed with and formed a coordination complex.

[0249] This hypothesis was further supported by Raman spectroscopy measurements: O=Os in Os(vi)-FeCN solution. VI The vibrational peak of =O was found to be shifted compared to Os(vi) alone (Figure 9b), indicating a change in the ligand of the Os(vi) molecule. The same shifted peak was also seen in the brain staining solution after the FeCN step (Figure 9c), indicating that FeCN during the sample staining process may have indeed reacted with Os(vi).

[0250] Taking these factors together, the possible functions of FeCN are - Diffuses through the sample and transports CN to exposed Os(vi). -The first step is to provide a complex with OsO2(OH)2(CN)2 (OsO4) (OsO2(OH)2(CN)2) that can be used to enhance membrane contrast. If the incubation of OsO4 before FeCN was not long enough to expose background Os(vi), then the FeCN incubation would only remove free Os(vi) in solution, not Os(vi) bound to cytoplasmic proteins, and thus membrane contrast would not be enhanced. If the incubation of OsO4 was long enough to expose protein-bound Os(vi), then the FeCN incubation could coordinate the exposed protein-bound Os(vi), removing Os(vi) from the sample and improving membrane contrast. The postulated form of this complex is OsO2(OH)2(CN)2 2- , OsO2(CN)4 2- (NPL23, NPL24) are possible.

[0251] This concept adds new insight into the long-standing chemistry of OsO4-FeCN film contrast enhancement (Fig. 9d): (1) OsO4 stained both membrane and cytoplasmic proteins (background) during short incubations; (2) upon prolonged incubation, OsO4 over-oxidized background proteins, thus exposing protein-bound Os(vi) to the aqueous phase; (3) FeCN transported CN(vi) throughout the sample. - and provides CN- to exposed protein-bound Os(vi) to form a stable coordination complex (this is Os(vi) and CN - Note that this is a compound between Os(vi) and FeCN, not between Os(vi) and FeCN itself as proposed in the 1970s (NPL36). - The coordination compounds between them can easily diffuse (NPL4).

[0252] Consideration of previous protocols within the framework of this exposure-based staining concept The concept of possible exposure modulation for membrane contrast enhancement would add surprising perspective to the findings of Karnovsky (NPL13) and de Bruijn's (NPL3) in the 1970s, who found FeCN as a stain enhancer: the reason this worked was because Fe 2+ CN in the coordination complex - is strong enough to maintain the - However, it did not react directly with the osmium compounds deposited in the membrane (as the osmium in the membrane is what we want to keep). In fact, the effect would be dramatic: the osmiated samples reacted with the free CN - When incubated in KCN, which provides CN, the entire sample would lose staining (NPL4). In contrast, FeCN is more strongly coordinated and will only bind CN when it encounters exposed / free Os(vi) in aqueous solution. - can be donated, and Os(vi) and CN - (NPL23, NPL24) that would be easily washed away (NPL4). This chemical concept was also consistent with earlier insights of "background washing" (NPL17, NPL22, NPL37).

[0253] The same incubation time-dependent effects would be present in the original rO protocol (Fig. 8e, f). The principles summarized above can be applied similarly: even if FeCN and OsO4 are mixed in rO (which would produce a mixture of OsO4, FeCN, FeCNiii, and Os(vi)), the FeCN-based reaction would not occur if background proteins were exposing Os(vi) to CN. - This will only occur when a complex is formed.

[0254] Providing a possible explanation for the staining gradient and precipitation zones (NPL11, NPL19) that appear when staining samples with rO of sizes larger than about 200 μm, the following was considered: the very slow centripetal diffusion of Os(vi) from all sides of the sample (Fig. 8c) generates a density of Os(vi) at a certain depth, which prevents CaC from stabilizing large amounts of Os(vi), and local disproportionation occurs (resulting in Os(vi)). IV O2 is produced, Fig. 8c, d).

[0255] To evaluate the rO (NPL3, NPL13), Hua (NPL11), and Mikula (NPL19) protocols in the framework of the exposure tuning concept, we investigated Os(vi) coordination reactions with various ligands (hydroxide, cacodylate, cyanide, etc., Fig. 9e). Coordination bonds from different ligands differ in their strength, which affects the stability of the complex. Similarly, different sizes of ligands result in different sizes of complexes, which affects the mobility of the complexes during diffusion. In water, Os(vi) coordinates with hydroxide ions (OH-) to form unstable complexes that are relatively easily disproportionated (NPL11, NPL15). In cacodylate buffer, Os(vi) is coordinated with cacodylate anion ((CH3)2AsO2 - ) possibly also via a coordination bond (NPL25), which is stronger and therefore inhibits the disproportionation of Os(vi) (Fig. 8d). The difference in the size of the coordination bonds between hydroxide and cacodylate may also contribute: in the hydroxide coordination, the distance is only two chemical bonds (Os-OH) whereas in the cacodylate coordination, the distance is four chemical bonds (Os-O-As-CH), which may affect the size and mobility of the final compound, possibly explaining why the [Os(vi)-cacodylate] complex diffuses more slowly (NPL11, NPL19).

[0256] It also explains why the Hua protocol (NPL11) works by separating OsO4 and FeCN, because by generating this separation locally it avoided long-distance Os(vi) diffusion; however, this is not essential, because Os(vi) does not contribute to membrane contrast and the OsO4-FeCN redox reaction leads to stain leakage, as shown in the text (Figure 6b, c).

[0257] In the case of formamide (NPL19) used in Mikula's protocol, Os(vi) coordinates with the amine group of formamide (NPL25). The coordination bond between formamide and Os(vi) results in three bond distances (Os-NCH), thus shorter than the four bond distances of cacodylic acid, which allows for mobility that enhances the mobility of the formamide coordination compound. The amine group of the ligand should also form a relatively stronger coordination bond than the hydroxide (NPL25), which would help prevent the precipitation discussed in "Reduced Osmium". However, the amine coordination bond may be even stronger than the cacodylic acid coordination bond, and thus may ultimately result in poor staining of the sample (similar to KCN). [Table 2-1] [Table 2-1] [Table 2-3]

[0258] Dehydration and resin infiltration of human samples was performed as follows:

[0259] Human samples were subjected to a graded ethanol dehydration step: 20% ethanol 30 min at 4 °C, 40% ethanol 30 min at 4 °C, 60% ethanol 30 min at 4 °C, 80% ethanol 30 min at 4 °C, 100% ethanol 45 min at room temperature. For resin infiltration, after three 45 min pure acetone washes, samples were subjected to graded Epon resin infiltration in acetone (for 10 ml resin: 5.9 g Epon medium, 2.25 g DDSA, 3.7 g MNA, 205 μl DMP) were all performed at 4°C: 12.5% ​​for 4 hours, 25% for 13 hours (overnight), 37.5% for 4 hours, 50% for 4 hours, 62.5% for 19 hours (overnight), 75% for 8 hours, 87.5% for 19 hours (overnight), 95% for 8 hours, 95% for 19 hours (overnight with 95% for the first time), 95% for 8 hours, 95% for 19 hours (overnight with 95% for the second time), 95% for 8 hours, 95% for 19 hours (overnight with 95% for the third time), 100% for 8 hours, 100% for 19 hours (overnight with 100% for the first time), 100% for 8 hours, 100% for 19 hours (overnight with 100% for the second time); the specimens were then embedded in freshly prepared Epon resin and cured at 60°C for 3 days.

[0260] In all solution exchange steps, the old solution was removed as much as possible to prevent interactions with the new solution. For samples with a size of 2-3 mm and brain hemisphere / whole brain samples, the extension of the above steps should be applied in a coordinated manner (meanwhile, the 2 ml Eppendorf tube should also be changed to a larger capacity tube). If any changes were made based on the currently recommended protocol, we also recommended checking the uniformity of staining with μCT after the main staining steps (i.e., 1st OsO4, FeCN, 2nd OsO4, Pg, 3rd OsO4, UA) to verify the effectiveness of the changes. [Table 3] [Table 4-1] [Table 4-2] [Table 4-3]

[0261] Surviving Artifacts In the large specimen protocol, three types of artifacts were observed: (1) blood vessels that were partially detached from the surrounding neuropil (Fig. 13b); (2) residual micro-breaks in the subcortical neuropil (Fig. 13d, e); and (3) damage to the outer parts of the cerebellum that may have been broken during the water incubation step (Fig. 13f).

[0262] To determine at which protocol step the vascular artifacts arose, we imaged 2 mm samples by μCT after 24 h of OsO4 incubation at room temperature; this method allowed us to avoid the effects of dehydration and resin infiltration. After the first OsO4 incubation step, the blood vessels were already detached (Figure 13c).

[0263] The same results were observed for samples incubated in OsO4 for 4 days at 4 °C (Figure 13c). While not a major concern for connectomics projects targeting synaptic circuits, further targeted optimization of the protocol seems necessary for projects in which full vascular integrity is essential.

[0264] As for the microbreaks in the subcortical regions, these were rather small (width ≤ 500 nm, see Fig. 13d) and difficult to detect by μCT. As they occurred infrequently and mainly in myelin-rich regions, neurite remodeling is expected to be largely unaffected.

[0265] As for the cerebellum damage artifact, it was evident from the staining process that it occurred in the first HO incubation step (Fig. 13g). This was found to be affected by the temperature of CaC incubation between OsO4 and FeCN: when the CaC incubation step was at 4°C, the cerebellum was preserved much better than when it was at room temperature (Fig. 13g).

[0266] Additional modification protocols for very fragile specimens The protocol steps in pure water generate large osmotic stresses, which can be a significant problem for large and fragile specimens. Although the addition of a second OsO4 step after FeCN stabilized the whole brain specimen near the Pg step (Figure 6i), incubation in pure water can still be problematic for some large specimens (e.g., specimens containing very large blood vessels from human brain). To accommodate such specimens, an alternative approach is to increase the osmotic pressure using chemically inert compounds. We found that CaC buffer does not function as an osmotic balancer during the Pg step, as too large an osmotic gradient (and breakage if the gradient is too strong) would be generated (Figure 6g).

[0267] One explanation for the appearance of such a gradient when incubated with CaC rather than HO during the Pg step is as follows (Fig. 6g, Fig. 10b): Because CaC can stabilize Os(vi) (Fig. 8d), there may be an interaction between Pg and the free Os(vi) stabilized by CaC in the sample. After the FeCN step, no free Os(vi) is expected to be present in the tissue (Fig. 9d); however, an additional second Os step may generate residual Os(vi). Indeed, when the second Os step was omitted, no staining gradient appeared during the Pg step, even with Pg-CaC interactions (Fig. 10a, c). On the other hand, the addition of KCl to HO, which is known to stabilize Os(vi) (NPL43), caused a staining gradient to appear during the Pg step (Fig. 10a, d); this could be reversed by temporarily adjusting the pH to 1 to remove Os(vi) from KCl-HO (which causes disproportionation of Os(vi), Fig. 10a, e).

[0268] In summary, our experiments support the conclusion that when using Pg, special care must be taken to avoid leaving free Os(vi) in the sample to avoid gradients (and their destruction if the gradients are too large) caused by Pg-Os(vi) interactions. This realization could, in principle, open new opportunities in the selection of incubation solutions before and after Pg.

[0269] For example, pilot experiments in which the second Os step was omitted (briefly, 1st Os → CaC → FeCN → CaC → Pg → CaC → Os HO / Os CaC; see Fig. 10a, c, f, g) represent an option for samples that are particularly sensitive to osmotic stress (e.g., mouse cerebellum or very large human cortical tissue samples).

[0270] The present invention can be described / summarized by the following items: [1] A method for staining a biological tissue sample, comprising the steps of: a) incubating the sample in a buffered aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% of a heavy metal compound; b) washing the sample in a buffered aqueous solution; c) incubating said sample in a buffered aqueous solution of 1.5-3.5%, preferably 2-3%, more preferably 2.2-2.75%, even more preferably 2.5% reducing agent; d) repeating step b); e) repeating step a); f) repeating step b); g) washing the sample with water; h) incubating said sample in an aqueous solution of 2.5-5.5%, preferably 3-5%, more preferably 3.5-4.5%, even more preferably 4% of a stain amplifier, preferably 1,2,3-trihydroxybenzene; i) repeating step g); j) repeating step a), wherein the heavy metal compound is dissolved in an aqueous solution; k) repeating step g); l) incubating said sample in an aqueous solution of 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4% of a heavy metal compound, said heavy metal compound being different from that in step a); m) further incubating the sample of step l), wherein the temperature is increased to at least 35° C.; and n) repeating step g).

[0271] [2] The method according to item [1], wherein the heavy metal compound in step a) is osmium tetroxide (OsO4), and the heavy metal compound in step l) is uranyl acetate (UA).

[0272] [3] The method according to item [1] or [2], wherein the reducing agent is ferrocyanide and / or ferricyanide.

[0273] [4] The method according to any one of items [1] to [3], wherein the incubation in the step a) is carried out at a temperature of 2 to 30° C. for 3 to 120 hours.

[0274] [5] The method according to any one of items [1] to [4], wherein the washing in the step b) is carried out at a temperature of less than 10° C., preferably 2 to 6° C., more preferably 3 to 5° C., and even more preferably 4° C., and wherein the washing is carried out for 0.5 to 190 hours.

[0275] [6] The method according to any one of items [1] to [5], wherein the incubation in step c) is carried out at a temperature below 10°C, preferably at 2 to 6°C, more preferably at 3 to 5°C, and even more preferably at 4°C, and wherein the incubation is carried out for 12 to 96 hours.

[0276] [7] The method according to any one of items [1] to [6], wherein the washing in the step d) is carried out at a temperature higher than 4° C., preferably 4 to 30° C., more preferably 15 to 25° C., and even more preferably 25° C., and wherein the washing is carried out for 0.5 to 120 hours.

[0277] [8] The method according to any one of items [1] to [7], wherein the incubation in step e) is carried out at a temperature higher than 4° C., preferably 4 to 30° C., more preferably 15 to 25° C., and even more preferably 25° C., and wherein the incubation is carried out for 1.5 to 72 hours.

[0278] [9] The method according to any one of items [1] to [8], wherein the washing in step f) is carried out at a temperature higher than 4° C., preferably 4 to 30° C., more preferably 15 to 25° C., even more preferably 25° C., and wherein the washing is carried out for 0.5 to 96 hours.

[0279]

[10] The method according to any one of items [1] to [9], wherein the washing in step g) is carried out at a temperature higher than 4° C., preferably 4 to 30° C., more preferably 15 to 25° C., even more preferably 25° C., and wherein the washing is carried out for 1 to 96 hours.

[0280]

[11] The method according to any one of items [1] to

[10] , wherein the incubation in step h) is carried out at a temperature higher than 4° C., preferably at 4 to 30° C., more preferably at 15 to 25° C., and even more preferably at 25° C., and wherein the incubation is carried out for 10 to 64 hours.

[0281]

[12] The method according to any one of items [1] to

[11] , wherein the washing in step i) is carried out at a temperature higher than 4° C., preferably 4 to 30° C., more preferably 15 to 25° C., even more preferably 25° C., and wherein the washing is carried out for 1 to 96 hours.

[0282]

[13] The method according to any one of items [1] to

[12] , wherein the incubation in step j) is carried out at a temperature higher than 4° C., preferably 4 to 30° C., more preferably 15 to 25° C., even more preferably 25° C., and wherein the incubation is carried out for 5 to 120 hours.

[0283]

[14] The method according to any one of items [1] to

[13] , wherein the washing in step k) is carried out at a temperature higher than 4° C., preferably 4 to 30° C., more preferably 15 to 25° C., even more preferably 25° C., and wherein the washing is carried out for 1 to 96 hours.

[0284]

[15] The method according to any one of items [1] to

[14] , wherein the incubation in step l) is carried out at a low temperature, preferably 2 to 6°C, more preferably 3 to 5°C, even more preferably 4°C, and wherein the incubation is carried out for 10 to 64 hours.

[0285]

[16] The method according to any one of items [1] to

[15] , wherein the incubation in step m) is carried out at 40 to 60°C, preferably 45 to 55°C, more preferably 50°C, and wherein the incubation is carried out for 1 to 6 hours.

[0286]

[17] The method according to any one of items [1] to

[16] , wherein the washing in step n) is carried out at a temperature higher than 4° C., preferably 4 to 30° C., more preferably 15 to 25° C., even more preferably 25° C., and wherein the washing is carried out for 1 to 96 hours.

[0287]

[18] The method according to any one of items [1] to

[17] , wherein step n) is followed by a step of dehydrating the sample (step o), preferably by incubating the sample in a graded ethanol series.

[0288]

[19] The method according to item

[18] , wherein the stepwise ethanol series ranges from 25 to 100%, preferably in steps of 25%, and wherein each step is carried out for 0.5 to 24 hours.

[0289]

[20] The method according to any one of items [1] to

[19] , wherein said step o) is followed by a step (step p) of incubating the sample in pure acetone at a temperature higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C.

[0290]

[21] The method according to item

[20] , wherein the incubation is carried out for 2 to 32 hours.

[0291] [21'] The method according to item

[20] , wherein the incubation is carried out for 2 hours to 3 days.

[0292]

[22] The method according to any one of items [1] to

[21] , wherein the step p) is followed by a step of embedding the sample using a low-viscosity epoxy resin infiltration method.

[0293]

[23] The method according to item

[22] , wherein the concentration of the epoxy resin is gradually increased.

[0294]

[24] The method according to any one of items [1] to

[23] , wherein the biological tissue sample is derived from nervous tissue.

[0295]

[25] The method according to any one of items [1] to

[24] , wherein the size of the biological tissue sample is 10 mm, 5 mm, and / or 2 to 3 mm.

[0296]

[26] The method according to any one of items [1] to

[25] , wherein the biological tissue sample is analyzed by electron microscopy, preferably three-dimensional electron microscopy.

[0297]

[27] The method according to any one of items [1] to

[26] , wherein the three-dimensional electron microscopy is preferably a high-throughput electron microscopy.

[0298]

[28] Use of the method according to any one of items [1] to

[27] in en bloc staining of mouse brain hemispheres, whole mouse brains, and cortical and / or subcortical volumes of higher mammals.

[0299]

[29] Use of the method according to any one of items [1] to

[28] in (high-density) neural circuit reconstruction or connectomics analysis, preferably high-resolution connectomics analysis.

[0300]

[30] Use of the method described in any one of items [1] to

[29] in the pathological examination of human nervous tissue samples.

[0301] [A1] A method for staining a biological tissue sample, comprising the following steps: Aa) incubating the sample in a buffered aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% of a heavy metal compound; Ab) washing the sample in a buffered aqueous solution; Ac) incubating the sample in a buffered aqueous solution of 1.5-3.5%, preferably 2-3%, more preferably 2.2-2.75%, even more preferably 2.5% reducing agent; Ad) washing the sample with the buffered aqueous solution or aqueous salt solution of step Ab), Ae) incubating said sample in an aqueous solution of 2.5-5.5%, preferably 3-5%, more preferably 3.5-4.5%, even more preferably 4% of a stain amplifier, preferably 1,2,3-trihydroxybenzene; Af) repeating step Ad), Ag) repeating step Aa), wherein said heavy metal compound is dissolved in an aqueous or buffered aqueous solution; Ah) washing the sample with an aqueous salt solution; Ai) incubating said sample in an aqueous salt solution of a heavy metal compound at 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4%, said heavy metal compound being different from that in step Aa), Aj) further incubating the sample of step Ai), wherein the temperature is increased to at least 35° C.; and Ak) repeating step Ah).

[0302] [A2] The method according to item [A1], wherein the heavy metal compound in step Aa) is osmium tetroxide (OsO4), and the heavy metal compound in step Ai) is uranyl acetate (UA).

[0303] [A3] The method according to item [A1] or [A2], wherein the reducing agent is ferrocyanide and / or ferricyanide.

[0304] [A4] The method according to any one of items [A1] to [A3], wherein the incubation in step Aa) is carried out at a temperature of 2 to 30°C for 3 to 120 hours.

[0305] [A5] The method according to any one of items [A1] to [A4], wherein the washing in step Ab) is carried out at a temperature below 10°C, preferably at 2 to 6°C, more preferably at 3 to 5°C, and even more preferably at 4°C, and wherein the washing is carried out for 0.5 to 190 hours.

[0306] [A6] The method according to any one of items [A1] to [A5], wherein the incubation in step Ac) is carried out at a temperature below 10°C, preferably at 2 to 6°C, more preferably at 3 to 5°C, even more preferably at 4°C, and wherein the incubation is carried out for 12 to 96 hours.

[0307] [A7] The method according to any one of items [A1] to [A6], wherein the washing in step Ad) is carried out at a temperature higher than 4°C, preferably 4 to 30°C, more preferably 15 to 25°C, even more preferably 25°C, and wherein the washing is carried out for 0.5 to 120 hours.

[0308] [A8] The method according to any one of items [A1] to [A7], wherein the incubation in step Ae) is carried out at a temperature higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C, and wherein the incubation is carried out for 10 to 64 hours.

[0309] [A9] The method according to any one of items [A1] to [A8], wherein the incubation in step Ag) is carried out at a temperature higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C, and wherein the incubation is carried out for 5 to 120 hours.

[0310] [A10] The method according to any one of items [A1] to [A9], wherein the aqueous salt solution is KCl or NaCl.

[0311] [A11] The method according to any one of items [A1] to [A10], wherein the washing in step Ah) is carried out at a temperature higher than 4°C, preferably 4 to 30°C, more preferably 15 to 25°C, even more preferably 25°C, and wherein the washing is carried out for 1 to 96 hours.

[0312] [A12] The method according to any one of items [A1] to [A11], wherein the incubation in step Ai) is carried out at a low temperature, preferably 2 to 6°C, more preferably 3 to 5°C, even more preferably 4°C, and wherein the incubation is carried out for 10 to 64 hours.

[0313] [A13] The method according to any one of items [A1] to [A12], wherein the incubation in step Aj) is carried out at 40 to 60°C, preferably 45 to 55°C, more preferably 50°C, and wherein the incubation is carried out for 1 to 6 hours.

[0314] [A14] The method according to any one of items [A1] to [A13], wherein the washing in step Ak) is carried out at a temperature higher than 4°C, preferably 4 to 30°C, more preferably 15 to 25°C, even more preferably 25°C, and wherein the washing is carried out for 1 to 96 hours.

[0315] [A15] The method according to any one of items [A1] to [A14], wherein step Ak) is followed by a step of dehydrating the sample (step A1), preferably by incubating the sample in a graded ethanol series.

[0316] [A16] The method according to item [A15], wherein the stepwise ethanol series ranges from 25 to 100%, preferably in steps of 25%, and wherein each step is carried out for 8 to 24 hours.

[0317] [A17] The method according to any one of items [A1] to [A16], wherein step A1) is followed by a step (step Am) of incubating the sample in pure acetone at a temperature higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C.

[0318] [A18] The method according to item [A17], wherein the incubation is carried out for 32 to 48 hours.

[0319] [A19] The method according to any one of items [A1] to [A18], wherein step Am) is followed by a step of embedding the sample using a low-viscosity epoxy resin infiltration method.

[0320] [A20] The method according to item [A19], wherein the concentration of the epoxy resin is gradually increased.

[0321] [A21] The method according to any one of items [A1] to [A20], wherein the biological tissue sample is derived from neural tissue, preferably selected from the group consisting of cerebellum and human cortical tissue samples.

[0322] [A22] The method according to item [A21], wherein the human cortical tissue sample has a size in a plane (base region) of 2-4 mm and a thickness of 2-5 mm, preferably wherein the sample has a size in a plane (base region) of 2-3 mm and a thickness of ≦2 mm.

[0323] [A23] The method according to any one of items [A1] to [A22], wherein the biological tissue sample is analyzed by electron microscopy, preferably three-dimensional electron microscopy.

[0324] [A24] The method according to item [A23], wherein the three-dimensional electron microscope is preferably a high-throughput electron microscope.

[0325] [A25] Use of the method according to any one of items [A1] to [A24] in en bloc staining of the cerebellum and / or cortex of higher mammals.

[0326] [A26] Use of the method according to any one of items [A1] to [A25] in (high-density) neural circuit reconstruction or connectomics analysis, preferably high-resolution connectomics analysis.

[0327] [A27] Use of the method described in any one of items [A1] to [A26] in the pathological examination of a human nervous tissue sample.

[0328] [A28] A method for staining a biological tissue sample, comprising the following steps: a) incubating the sample in a buffered aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% of a heavy metal compound; b) washing the sample in a buffered aqueous solution; c) incubating said sample in a buffered aqueous solution of 1.5-3.5%, preferably 2-3%, more preferably 2.2-2.75%, even more preferably 2.5% reducing agent; d) repeating step a); e) repeating step b); f) washing the sample in water; g) incubating said sample in an aqueous solution of 2.5-5.5%, preferably 3-5%, more preferably 3.5-4.5%, even more preferably 4% of a stain amplifier, preferably 1,2,3-trihydroxybenzene; h) repeating step f); i) repeating step a), wherein said heavy metal compound is dissolved in an aqueous solution; j) repeating step g); k) incubating said sample in an aqueous solution of 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4% of a heavy metal compound, wherein said heavy metal compound is different from that of step a), l) further incubating the sample of step k), wherein the temperature is increased to at least 35° C.; and m) repeating step f).

[0329] [A29] The method according to item [A28], wherein the heavy metal in step a) is osmium tetroxide (OsO4), and the heavy metal in step k) is uranyl acetate (UA).

[0330] [A30] The method according to item [A28] or [A29], wherein the reducing agent is ferrocyanide and / or ferricyanide.

[0331] [A31] The method according to any one of items [A28] to [A30], wherein the incubation in step a) is carried out at a temperature of 2 to 30°C for 3 to 120 hours.

[0332] [A32] The method according to any one of Items [A28] to [A31], wherein the washing in step b) is carried out at a temperature of less than 10°C, preferably 2 to 6°C, more preferably 3 to 5°C, and even more preferably 4°C, and wherein the washing is carried out for 0.5 to 190 hours.

[0333] [A33] The method according to any one of [A28] to [A32], wherein the incubation in step c) is carried out at a temperature below 10°C, preferably at 2 to 6°C, more preferably at 3 to 5°C, and even more preferably at 4°C, and wherein the incubation is carried out for 12 to 96 hours.

[0334] [A34] The method according to any one of items [A28] to [A33], wherein the incubation in step k) is carried out at a low temperature, preferably 2 to 6°C, more preferably 3 to 5°C, even more preferably 4°C, and wherein the incubation is carried out for 10 to 64 hours, and wherein the incubation in step l) is carried out at 40 to 60°C, preferably 45 to 55°C, more preferably 50°C, and wherein the incubation is carried out for 1 to 6 hours.

[0335] [A35] The method according to any one of items [A28] to [A34], wherein step m) is followed by a step (step n)) of dehydrating the sample, preferably by incubating the sample in a graded ethanol series.

[0336] [A36] The method according to any one of items [A28] to [A35], wherein step n) is followed by a step (step o) of incubating the sample in pure acetone at a temperature higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C.

[0337] [A37] The method according to any one of items [A28] to [A36], wherein step o) is followed by a step of embedding the sample using a low-viscosity epoxy resin infiltration method, preferably a low-viscosity epoxy resin infiltration method using a gradually increasing concentration of epoxy resin.

[0338] [A38] The method according to any one of items [A28] to [A37], wherein the biological tissue sample is derived from nervous tissue, preferably human nervous tissue.

[0339] [A39] The method according to any one of items [A28] to [A38], wherein the size of the biological tissue sample is 2-4 mm in planar surface (base region) and 2-5 mm in thickness, preferably the size of the sample is 2-3 mm in planar surface (base region) and ≦2 mm in thickness.

[0340] [A40] The method according to any one of items [A28] to [A39], wherein the biological tissue sample is analyzed by electron microscopy, preferably a three-dimensional electron microscope, more preferably a high-throughput electron microscope.

[0341] [A41] Use of the method according to any one of items [A28] to [A40] in en bloc staining of mouse brain hemispheres, whole mouse brains, and cortical and / or subcortical volumes of higher mammals.

[0342] [A42] Use of the method according to any one of items [A28] to [A41] in (high-density) neural circuit reconstruction or connectomics analysis, preferably high-resolution connectomics analysis.

[0343] Cited Non-Patent Literature [CrossRef] Abbott, LF, DD Bock, EM Callaway, W Denk, C Dulac, AL Fairhall, I Fiete, KM Harris, M Helmstaedter, V Jain, N Kasthuri, Y LeCun, JW Lichtman, PB Littlewood, L Luo, JHR Maunsell, RC Reid, Rosen BR, Rubin GM, Sejnowski TJ, Seung HS, Svoboda K, Tank DW, Tsao D and Van Essen DC (2020). "The Mind of a Mouse." Cell 182(6); 1372-1 [CrossRef] Briggman, KL, M. Helmstaedter and W. Denk (2011). "Wiring specificity in the direction-selectivity circuit of the retina." Nature 471: 183-190. [Chapter 3] Bruijn, WC d. (1973). "Glycogen, Its Chemistry and Morphologic Appearance in the Electron Microscope I. A Modified OsO4fixative Which Selectively Contrasts Glycogen." J. Ultrastructure Research 50 . [Non-Patent Document 4] Bruijn, WCD, & Breejen, PD (1975). Glycogen, its chemistry and morphological appearance in the electron microscope. II. The complex formed in the selective contrast staining of glycogen. Histochemical Journal, 7, 205-229. [Non-Patent Document 5] Denk, W. and H. Horstmann (2004). "Serial block-face scanning electron microscopy to reconstruct three-dimensional tissue nanostructure." PLoS Biol 2(11): e329. [Non-patent document 6] Griffith, WP (1962). Cyanide complexes of the transition metals. Quarterly Reviews, Chemical Society, 16, 188-207. doi: 10.1039 / qr9621600188 [Non-Patent Document 7] Hayat, MA (1981). "Fixation for Electron Microscopy." [Non-Patent Document 8] Hayworth, KJ, N. Kasthuri, R. Schalek and JW Lichtman (2006). "Automating the Collection of Ultrathin Serial Sections for Large Volume TEM Reconstructions." Microscopy and Microanalysis 12(S02): 86-87. [CrossRef] Hayworth, KJ, D. Peale, M. Januszewski, GW Knott, Z. Lu, CS Xu and HF Hess (2020). "Gas cluster ion beam SEM for imaging of large tissue samples with 10 nm isotropic resolution." Nature Methods 17: 68-71. [CrossRef] Holcomb, PS, Hoffpauir BK, Hoyson MC, Jackson DR, Deerinck TJ, Marrs GS, Dehoff M, Wu J, Ellisman MH and Spirou GA (2013). "Synaptic inputs compete during rapid formation of the calyx of held: A new model system for neural development." Journal of Neuroscience 33: 12954–12969. [CrossRef] Hua, Y., P. Laserstein and M. Helmstaedter (2015). "Large-volume en-bloc staining for electron microscopy-based connectomics." Nature Communications 6: 1-7. [CrossRef]12]Januszewski, M., J. Kornfeld, PH Li, A. Pope, T. Blakely, L. Lindsey, J. Maitin-Shepard, M. Tyka, W. Denk and V. Jain (2018). "High-precision automated reconstruction of neurons with flood-filling networks." Nat Methods 15(8): 605-610. [Non-Patent Document 13] Karnovsky, M. J. (1971). Use of ferrocyanide-reduced osmium tetroxide in electron microscopy. 11th annual meeting, the American Society for Cell Biology. [Non-Patent Document 14] Kazmierski, T. M. and P. F. Millington (1979). "Modification of an SEM for backscattered electron imaging at low vacuum." Journal of Microscopy 117: 431-436. [Non-Patent Document 15] Korn, E. D. (1967). A chromatographic and spectrophotometric study of the products of the reaction of osmium tetroxide with unsaturated lipids. The Journal of cell biology, 34, 627-638. doi: 10.1083 / jcb.34.2.627 [Non-Patent Document 16] Kuan, A. T., J. S. Phelps, L. A. Thomas, T. M. Nguyen, J. Han, C. L. Chen, A. W. Azevedo, J. C. Tuthill, J. Funke, P. Cloetens, A. Pacureanu and W. C. A. Lee (2020). "Dense neuronal reconstruction through X-ray holographic nano-tomography." Nature Neuroscience 23: 1637-1643. [Non-Patent Document 17] Litman, RB, & Barrnett, RJ (1972). The mechanism of the fixation of tissue components by osmium tetroxide via hydrogen bonding. Journal of Ultrasructure Research, 38, 63-86. doi: 10.1016 / S0022-5320(72)90084-6 [Non-Patent Document 18] Loos, MR, LAF Coelho, SH Pezzin and SC Amico (2008). "The effect of acetone addition on the properties of epoxy." Polimeros 18: 76-80. [Non-Patent Document 19] Mikula, S. and W. Denk (2015). "High-resolution whole-brain staining for electron microscopic circuit reconstruction." Nature Methods 12: 541-546. [Non-Patent Document 20] Motta, A., M. Berning, KM Boergens, B. Staffler, M. Beining, S. Loomba, P. Hennig, H. Wissler and M. Helmstaedter (2019). "Dense connectomic reconstruction in layer 4 of the somatosensory cortex." Science 366. [Non-Patent Document 21] Motta, A., M. Schurr, B. Staffler and M. Helmstaedter (2019). "Big data in nanoscale connectomics, and the greed for training labels." Curr Opin Neurobiol 55: 180-187. [Non-Patent Document 22] Neiss, WF (1984). Electron staining of the cell surface coat by osmium-low ferrocyanide *. Histochemistry, 231-242. [Non-Patent Document 23] Opekar, F., & Beran, P. (1977). STUDY OF THE CYANIDE COMPLEXES THE REDUCTION THE CYANIDE COMPLEX OF Os (V1) ON A PLATINUM rde. Electrochimica Acta, 22, 243-248. [Non-Patent Document 24] Opekar, F., & Beran, P. (1977). Voltammetric study of the cyanide complexes of osmium-II. The behavior of lower oxidation state cyanide complexes of osmium on a platinum rde. Electrochimica Acta, 22, 249-254. doi: 10.1016 / 0013-4686(77)85069-X [Non-patent document 25] Osmium: Inorganic & Coordination Chemistry (2011). [Non-Patent Document 26] Palade, GE (1952). “A Study of Fixation for Electron Microscopy.” The Journal of experimental medicine, 95(3), 285-298. [Non-Patent Document 27] Porter, K. R., & Kallman, F. (1953). The properties and effects of osmium tetroxide as a tissue fixative with special reference to its use for electron microscopy. Experimental Cell Research, 4, 127-141. doi: 10.1016 / 0014-4827(53)90195-5 [Non-Patent Document 28] Riemersma, J. C., Alsbach, E. J. J., & Bruijn, W. C. D. (1984). Chemical aspects of glycogen contrast-staining by potassium osmate. Histochemical Journal, 136, 123-136. [Non-Patent Document 29] Seligman, A. M., H. L. Wasserkrug and J. S. Hanker (1966). "A new staining method (OTO) for enhancing contrast of lipid--containing membranes and droplets in osmium tetroxide--fixed tissue with osmiophilic thiocarbohydrazide (TCH)." Journal of Cell Biology 30: 424-432. [Non-Patent Document 30]Sommons, R. and P. Marquis (1997). "Application of the low vacuum scanning electron microscope to the study of biomaterials and mammalian cells." Biomaterials 18: 81-86. [Non-Patent Document 31] Stroh, S., E. W. Hammerschmith, D. W. Tank, H. S. Seung and A. Adrian (2021). "In situ X-ray assisted electron microscopy staining for large biological samples." bioRxiv. [Non-Patent Document 32] Tirler, A. O., Persson, I., Hofer, T. S., & Rode, B. M. (2015). Is the Hexacyanoferrate(II) Anion Stable in Aqueous Solution? A Combined Theoretical and Experimental Study. Inorganic Chemistry, 54, 10335-10341. doi: 10.1021 / acs.inorgchem.5b01701 [Non-Patent Document 33] Vidil, T., F. Tournilhac, S. Musso, A. Robisson, T. Vidil, F. Tournilhac, S. Musso, A. Robisson and L. Leibler (2016). "Control of reactions and network structures of epoxy thermosets." Progress in Polymer Science 62: 126-179. [Non-Patent Document 34] Walton, J. (1979). "Lead Aspartate, an En Bloc Contrast Stain Particularly Useful for Ultrastructural Enzymology." The Joural of Histochemistry and Cytochemistry 27(10): 1337-1342. [Non-Patent Document 35] Watson, M. L. (1958). “Staining of Tissue Sections for Electron Microscopy with Heavy Metals.” The Journal of Biophysical and Biochemical Cytology, 4(4), 475. [Non-Patent Document 36] White, D. L., Mazurkiewicz, J. E., & Barrnett, R. J. (1979). A chemical Mechanism for Tissue Staining by Osmium Tetroxide-Ferrocyanide Mixtures. The Journal of Histochemistry and Cytochemistry, 1084-1091. [Non-Patent Document 37] Willingham, C. and A. Rutherford (1984). "Use of Osmium-Thiocarbohydrazide-Osmium and Ferrocyanide-reduced Methods Contrast and Preservation in to Enhance Membrane Contrast and Preservation in Cultured Cells." The Journal of Histochemistry and Cytochemistry 32: 455-460. [Non-Patent Document 38] Yin, W., Brittain, D., Borseth, J., Scott, M. E., Williams, D., Perkins, J.,... & da Costa, N. M. (2020). “A petascale automated imaging pipeline for mapping neuronal circuits with high-throughput transmission electron microscopy”. Nature communications, 11(1), 1-12.). [Google Scholar 39]Arganda-Carreras , I. , Kaynig , V. , Rueden , C. , Eliceiri , KW , Schindelin , J. , Cardona , A. , & Sebastian Seung , H. (2017). Trainable Weka Segmentation: a machine learning tool for microscopy pixel classification. Bioinformatics, 33(15), 2424-2426 [CrossRef] 40] Loomba, S., Straehle, J., Gangadharan, V., Heike, N., Khalifa, A., Motta, A., ... & Helmstaedter, M. (2022). “Connectomic comparison of mouse and human cortex”. Science, 377(6602), p0924. [CrossRef] Karimi , A. , Odenthal , J. , Drawitsch , F. , Boergens , K. M. , & Helmstaedter , M. (2020). “Cell-type specific innervation of cortical pyramidal cells at their apical dendrites”. Elife, 9, e46876. [Google Scholar 42] Boergens , KM , Berning , M. , Bocklisch , T. , Braunlein , D. , Drawitsch , F. , Frohnhofen , J. , … & Helmstaedter , M. (2017). “webKnossos: efficient online 3D data annotation for connectomics”. Nature Methods, 14(7), 691-694. [Non-Patent Document 43] Collinson, SR, & Schroder, M. (2011). Osmium: Inorganic & coordination chemistry. Encyclopedia of Inorganic and Bioinorganic Chemistry.

[0344] All non-patent literature cited herein is hereby incorporated by reference in its entirety.

Claims

1. A method for staining a biological tissue sample, comprising the steps of: a) incubating said sample in a buffered aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% heavy metal compound; b) washing the sample in a buffered aqueous solution; c) incubating said sample in a buffered aqueous solution of 1.5-3.5%, preferably 2-3%, more preferably 2.2-2.75%, even more preferably 2.5% reducing agent; d) repeating step b); e) repeating step a); f) repeating step b); g) washing the sample with water; h) incubating said sample in an aqueous solution of 2.5-5.5%, preferably 3-5%, more preferably 3.5-4.5%, even more preferably 4% of a stain amplifier, preferably 1,2,3-trihydroxybenzene; i) repeating step g); j) repeating step a), wherein the heavy metal compound is dissolved in an aqueous solution; k) repeating step g); l) incubating said sample in an aqueous solution of a heavy metal compound at 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4%, said heavy metal compound being different from that of step a); m) further incubating the sample of step l), wherein the temperature is raised to at least 35°C; and n) repeating step g).

2. The heavy metal compound in step a) is osmium tetroxide (OsO ４ 2. The method of claim 1, wherein the heavy metal compound in step l) is uranyl acetate (UA).

3. 2. The method of claim 1, wherein the reducing agent is ferrocyanide and / or ferricyanide.

4. 2. The method of claim 1, wherein the incubation in step a) is carried out at a temperature of 2 to 30° C. for 3 to 120 hours.

5. 2. The method of claim 1, wherein the washing of step b) is carried out at a temperature below 10°C, preferably 2-6°C, more preferably 3-5°C, even more preferably 4°C, and wherein said washing is carried out for 0.5-190 hours.

6. 2. The method of claim 1, wherein the incubation of step c) is carried out at a temperature below 10°C, preferably between 2 and 6°C, more preferably between 3 and 5°C, even more preferably at 4°C, and wherein said incubation is carried out for 12 to 96 hours.

7. 2. The method of claim 1, wherein the incubation in step l) is carried out at low temperature, preferably 2-6°C, more preferably 3-5°C, even more preferably 4°C, and wherein said incubation is carried out for 10-64 hours, and wherein the incubation in step m) is carried out at 40-60°C, preferably 45-55°C, even more preferably 50°C, and wherein said incubation is carried out for 1-6 hours.

8. 2. The method of claim 1, wherein after step n) the sample is dehydrated, preferably by incubating the sample in a graded ethanol series.

9. 2. The method of claim 1, wherein after step o) the sample is incubated in pure acetone at a temperature above 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C.

10. 2. The method according to claim 1, wherein after step p), the sample is embedded by using a low-viscosity epoxy resin infiltration method, preferably a low-viscosity epoxy resin infiltration method in which the concentration of the epoxy resin is gradually increased.

11. The method of claim 1 , wherein the biological tissue sample is derived from neural tissue.

12. The method of claim 1, wherein the size of the biological tissue sample is 10 mm, 5 mm, and / or 2-3 mm.

13. The method of claim 1, wherein the biological tissue sample is analyzed by electron microscopy, preferably three-dimensional electron microscopy, more preferably high-throughput electron microscopy.

14. Use of the method according to any one of claims 1 to 13 for en bloc staining of mouse brain hemispheres, whole mouse brains, cortical and / or subcortical volumes of higher mammals.

15. Use of the method according to any one of claims 1 to 13 in (high density) neural circuit reconstruction or connectomics analysis, preferably in high resolution connectomics analysis.

16. A method for staining a biological tissue sample, comprising the steps of: Aa) incubating the sample in a buffered aqueous solution of 1-3%, preferably 1.5-2.5%, more preferably 1.8-2.2%, even more preferably 2% of a heavy metal compound; Ab) washing the sample in a buffered aqueous solution; Ac) incubating said sample in a buffered aqueous solution of 1.5-3.5%, preferably 2-3%, more preferably 2.2-2.75%, even more preferably 2.5% reducing agent; Ad) washing the sample with the buffered aqueous solution or aqueous salt solution of step Ab); Ae) incubating said sample in an aqueous solution of 2.5-5.5%, preferably 3-5%, more preferably 3.5-4.5%, even more preferably 4% dye amplifier, preferably 1,2,3-trihydroxybenzene; Af) repeating step Ad); Ag) repeating step Aa), wherein the heavy metal compound is dissolved in an aqueous or buffered aqueous solution; Ah) washing the sample with an aqueous salt solution; Ai) incubating said sample in an aqueous salt solution of a heavy metal compound at 3-5%, preferably 3.5-4.5%, more preferably 3.75-4.25%, even more preferably 4%, wherein said heavy metal compound is different from that in step Aa); Aj) further incubating the sample of step Ai), wherein the temperature is increased to at least 35°C; and Ak) Repeating step Ah).

17. The heavy metal compound in step Aa) is osmium tetroxide (OsO ４ 17. The method of claim 16, wherein the heavy metal compound of step Ai) is uranyl acetate (UA).

18. 17. The method of claim 16, wherein the reducing agent is ferrocyanide and / or ferricyanide.

19. 17. The method of claim 16, wherein the incubation in step Aa) is carried out at a temperature of 2 to 30° C. for 3 to 120 hours.

20. 17. The method according to claim 16, wherein the washing of step Ab) is carried out at a temperature below 10°C, preferably at 2-6°C, more preferably at 3-5°C, even more preferably at 4°C, and wherein said washing is carried out for 0.5-190 hours.

21. 17. The method according to claim 16, wherein the incubation of step Ac) is carried out at a temperature below 10°C, preferably at 2-6°C, more preferably at 3-5°C, even more preferably at 4°C, and wherein said incubation is carried out for 12-96 hours.

22. 17. The method according to claim 16, wherein the washing of step Ad) is carried out at a temperature higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C, and wherein said washing is carried out for a time period of 0.5 to 120 hours.

23. 17. The method of claim 16, wherein the incubation of step Ae) is carried out at a temperature higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C, and wherein said incubation is carried out for 10 to 64 hours.

24. 17. The method of claim 16, wherein the incubation of step Ag) is carried out at a temperature higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C, and wherein said incubation is carried out for 5 to 120 hours.

25. 17. The method of claim 16, wherein the aqueous salt solution is KCl or NaCl.

26. 17. The method of claim 16, wherein the washing of step Ah) is carried out at a temperature above 4°C, preferably 4 to 30°C, more preferably 15 to 25°C, even more preferably 25°C, and wherein said washing is carried out for 1 to 96 hours.

27. 17. The method of claim 16, wherein the incubation of step Ai) is carried out at low temperature, preferably 2-6°C, more preferably 3-5°C, even more preferably 4°C, and wherein said incubation is carried out for 10-64 hours.

28. 17. The method of claim 16, wherein the incubation of step Aj) is carried out at 40-60°C, preferably 45-55°C, more preferably 50°C, and wherein said incubation is carried out for 1-6 hours.

29. 17. The method according to claim 16, wherein the washing of step Ak) is carried out at a temperature higher than 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C, and wherein said washing is carried out for 1 to 96 hours.

30. 17. The method according to claim 16, wherein after step Ak) the sample is dehydrated, preferably by incubating the sample in a graded ethanol series.

31. 31. The method of claim 30, wherein the stepwise ethanol series ranges from 25 to 100%, preferably varying in steps of 25%, and wherein each step is carried out for 8 to 24 hours.

32. 17. The method according to claim 16, wherein after step A1) the sample is incubated in pure acetone at a temperature above 4°C, preferably between 4 and 30°C, more preferably between 15 and 25°C, even more preferably at 25°C (step Am)).

33. 33. The method of claim 32, wherein the incubation is for 32 to 48 hours.

34. 17. The method according to claim 16, wherein after step Am), the sample is embedded by using a low viscosity epoxy resin infiltration method.

35. 35. The method of claim 34, wherein the concentration of the epoxy resin is gradually increased.

36. 17. The method of claim 16, wherein said biological tissue sample is derived from neural tissue, preferably selected from the group consisting of cerebellum and human cortical tissue samples.

37. 37. The method of claim 36, wherein the human cortical tissue sample has a size of 2-4 mm in planar (base region) and 2-5 mm in thickness, preferably a size of 2-3 mm in planar (base region) and a thickness of ≦2 mm.

38. 17. The method of claim 16, wherein the biological tissue sample is analyzed by electron microscopy, preferably three-dimensional electron microscopy.

39. 39. The method of claim 38, wherein said three-dimensional electron microscopy is preferably high-throughput electron microscopy.

40. Use of the method according to any one of claims 16 to 39 in en bloc staining of the cerebellum and / or cortex of higher mammals.

41. Use of the method according to any one of claims 16 to 39 in (high density) neural circuit reconstruction or connectomics analysis, preferably in high resolution connectomics analysis.

42. Use of the method according to any one of claims 16 to 39 in the pathological examination of human nervous tissue samples.

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