A surface fluorescent tomographic resin embedding method suitable for immunostaining samples

Abstract

The application discloses a surface fluorescence chromatography resin embedding method suitable for immunostaining samples, and comprises the following steps: S1, biological tissue sample pretreatment; S2, gradient ethanol dehydration and tissue blackening; S3, gradient resin permeation; S4, acidification treatment; and S5, resin polymerization molding. The application also comprises a biological tissue sample imaging method, which comprises the step of imaging the biological tissue sample obtained by the embedding method in a fMOST system sodium carbonate solution environment. The application temporarily quenches the pH-sensitive chemical probe by adding HAc; in combination with the fMOST imaging system, the reactivation is realized in the alkaline solution environment, the blackening chemical chromatography high-precision imaging is realized by reactivating the sample imaging surface, and the immunostaining sample has the advantages of high fluorescence retention rate.

Description

Technical Field

[0001] This invention belongs to the field of resin embedding technology, and more specifically, relates to a method for embedding surface fluorescence chromatography resin in immunostained samples. Background Technology

[0002] Immunostaining techniques are key tools in research fields such as neurobiology. In recent years, large-volume immunofluorescence staining techniques, represented by iDISCO, have significantly improved the ability to stain whole tissues at the centimeter-level organ level. Combined with large-volume imaging techniques such as light sheet imaging (LSFM) and optical microscopy section tomography (fMOST), systematic imaging of specific molecules and fine structures in intact tissues can be achieved.

[0003] However, traditional light-sheet imaging has relatively low spatial resolution, making it difficult to directly meet the needs of fine structure imaging. Microscopic optical section tomography (fMOST), with its high imaging resolution, is a potentially superior solution for fine structure and molecular imaging, but this technique relies on resin embedding of the sample. Acrylic resins, such as GMA, HM20, and LR White, have been widely used in fMOST imaging due to their excellent mechanical strength and optical transparency. However, existing embedding methods are mainly developed for fluorescent protein-labeled samples, and their application to immunofluorescence-stained samples has significant limitations. Specifically, the signal in immunofluorescence staining originates from chemical probes, facing two challenges: first, the fluorescent signal of chemical probes is easily bleached by excitation light during imaging, leading to signal attenuation; second, because immunofluorescence staining simultaneously labels all specific molecules in the tissue, it cannot achieve sparse labeling like fluorescent protein labeling, resulting in dense signals and significant signal interference in the out-of-focus lower layers during imaging, severely affecting image quality and subsequent analysis.

[0004] Therefore, this invention develops a tissue blackening and chemical chromatography resin embedding reagent and method suitable for fine imaging of immunofluorescence stained samples, which has important application value. Summary of the Invention

[0005] To address the shortcomings of existing technologies and practical needs, this invention provides a surface fluorescence chromatography resin embedding method suitable for immunostained samples, and an imaging method for biological tissue samples obtained by this embedding method.

[0006] Specifically, the present invention adopts the following technical solution:

[0007] In a first aspect, the present invention provides a method for embedding surface fluorescent chromatography resin in immunostained samples, the method comprising the following steps:

[0008] S1. Pretreatment of biological tissue samples: Fix and label biological tissue samples with fluorescent probes for later use;

[0009] S2, Gradient ethanol dehydration and tissue blackening: The pretreated biological tissue samples were dehydrated by a gradient of different concentrations of ethanol, and then transferred to SBB / EtOH solution for dehydration overnight at 4°C.

[0010] S3, Gradient Resin Permeation: The dehydrated biological tissue sample is permeated with a gradient of resin / ethanol at different concentrations, then transferred to 100% resin for permeation, the solution is changed and left overnight, and then the 100% resin is replaced and incubated the next day.

[0011] S4. Acidification treatment: Transfer the fully permeated biological tissue sample into the acidification resin solution and treat at 4°C.

[0012] S5. Resin Polymerization Molding: The acidified biological tissue sample is placed in a gelatin capsule, and SBB / ABVN / resin solution is added. Thermal polymerization is then carried out to complete the embedding of the biological tissue sample.

[0013] In some implementations, the biological tissue sample in step S1 is mouse brain or mouse peripheral organ tissue.

[0014] In some implementations, in step S1, the biological tissue sample is fixed with 4% PFA solution and then rinsed multiple times with PBS buffer.

[0015] In some implementations, in step S1, the biological tissue sample is fluorescently labeled using a FITC fluorescent probe, and then washed multiple times with PBS buffer.

[0016] In some implementations, in step S2, the pretreated biological tissue sample is sequentially dehydrated with 50%, 75%, and 95-100% ethanol in a gradient.

[0017] In some implementations, step S2 involves dehydration for 1-3 hours at different concentrations of ethanol gradients.

[0018] In some embodiments, the SBB / EtOH solution in step S2 is obtained by adding 0.5-0.6% SBB to 95-100% ethanol and then stirring to dissolve it.

[0019] In this invention, gradient alcohol is used to replace water within the tissue, preventing drastic sample shrinkage caused by direct dehydration from high-concentration alcohol. Simultaneously, pre-osmosis with SBB (sulfate-based ethanol) establishes a preliminary background inhibition environment.

[0020] In some implementations, in step S3, the biological tissue sample is permeated sequentially with 50% resin / EtOH and 75% resin / EtOH.

[0021] In some implementations, in step S3, the resin / EtOH gradients at different concentrations are permeated for 2-3 hours each.

[0022] In some implementations, step S3 involves transferring the gradient-permeable biological tissue sample into 100% resin for 1-3 hours.

[0023] In some implementations, in step S3, the incubation is carried out for 24-48 hours the following day in a fresh 100% resin solution.

[0024] In some embodiments, the resin mentioned in step S3 is an acrylic resin, such as HM20 resin, GMA resin, or BMA resin.

[0025] In this invention, infiltration is a slow physical diffusion process. If infiltration is insufficient, the monomer density will be uneven between the center and the edge of the tissue, which may lead to sample deformation or cavitation (bursting polymerization) after aggregation. The infiltration time can be determined according to the size of the tissue block. For example, 2 hours of infiltration into the whole brain of young mice is sufficient, while 3 hours of infiltration into the liver of adult mice is sufficient.

[0026] In some embodiments, the acidified resin solution in step S4 is obtained by adding 0.05-0.1% acetic acid to 100% resin mother liquor.

[0027] In some implementations, step S4 involves treating the biological tissue sample in an acidified resin solution for 12-15 hours.

[0028] In some embodiments, the resin in step S4 is an acrylic resin, such as HM20 resin, GMA resin, or BMA resin.

[0029] In this invention, insufficient acidification time (<12 hours) prevents the acidic components from fully diffusing into the deeper layers of the sample, making it difficult to effectively reactivate the fluorescence signal and resulting in low imaging brightness. Excessive acidification or excessively long acidification time can lead to excessively low local pH, which in turn directly damages the molecular structure of the fluorophore, causing irreversible fluorescence quenching.

[0030] In some embodiments, the SBB / ABVN / resin solution in step S5 is obtained by adding 0.5-0.6% SBB and 0.45-0.8% ABVN to the resin and stirring until homogeneous.

[0031] In some embodiments, the resin mentioned in step S5 is an acrylic resin, such as HM20 resin, GMA resin, or BMA resin.

[0032] In some implementations, the thermal polymerization described in step S5 is carried out in a closed, constant-temperature and constant-pressure oven.

[0033] In some implementations, the temperature of the thermal polymerization in step S5 is 38-50°C, and the time of the thermal polymerization is 6-24 hours.

[0034] In some implementations, the thermal polymerization conditions in step S5 are 38°C for 4 hours and 40°C for 8 hours.

[0035] In this invention, if the polymerization temperature is below 38°C, the reaction kinetic energy is insufficient, making it difficult for the monomer to initiate polymerization or resulting in incomplete polymerization. This leads to the embedding block being too soft and unable to be physically sliced. If the polymerization temperature is too high, the polymerization reaction is too exothermic, which can cause a "burst polymerization" phenomenon, resulting in a large number of bubbles or cracks inside the sample, directly damaging the biological sample.

[0036] In a second aspect, the present invention provides an imaging method for biological tissue samples, the method comprising the step of imaging a biological tissue sample obtained by any of the embedding methods of the present invention in a sodium carbonate solution environment of an fMOST system.

[0037] In some embodiments, the concentration of the sodium carbonate solution is 5.5-6 g / L.

[0038] In some implementations, PI real-time staining is combined during the imaging process to obtain the structural information of biological tissue samples.

[0039] Compared with the prior art, the present invention has the following advantages:

[0040] (1) The present invention adds SBB background inhibitors, which enables long-term imaging of samples stained with acrylic resins such as HM20, BMA, and GMA without quenching.

[0041] (2) Based on the immunostaining of FITC samples, the present invention adds a chemical reactivation process: by adding HAc, the pH-sensitive chemical probe is temporarily quenched; combined with the fMOST imaging system, imaging is performed in an alkaline environment to achieve reactivation, and the sample imaging surface is reactivated to achieve high-precision imaging of blackened chemical chromatography, which has the advantage of high fluorescence retention rate for immunostaining samples.

[0042] (3) The embedding method described in this invention is used to embed biological tissues. The tissues are uniform in texture, free of bubbles and cracks, and the slicing process is smooth. During the experiment, the SBB background inhibitor and chemical chromatography have a superimposed effect: by introducing HAc to selectively quench the FITC signal outside the focal plane, the layer-by-layer signal resolution effect of optical chromatography is simulated at the chemical level, which effectively suppresses the interference of the out-of-focus signal on the current imaging layer, so that the FITC signal of the target layer can be clearly highlighted. With the background suppression effect of SBB, the imaging background interference is significantly reduced, so that the FITC fluorescence signal is stable and bright, the imaging is clear, and the fine structure inside the tissue can be clearly observed. If PI staining solution is added, the cell distribution, tissue boundary and other structural information of the tissue can be displayed in real time through its specific binding with cell DNA, which further assists in interpreting the localization of FITC signal. If PI staining solution is omitted, FITC signal imaging alone can still meet the core requirements of high-resolution imaging of fMOST system.

[0043] (4) In this embodiment, the chemical chromatography selectively quenches the FITC fluorescence signal in the out-of-focus layer using chemical reagents, thereby completing the pre-suppression of the out-of-focus background during the sample preparation stage. This, in conjunction with the background suppression effect of SBB, further improves the stability and signal-to-noise ratio of the FITC signal, highlighting the technical advantages of this invention. Attached Figure Description

[0044] Figure 1 This is a comparison of the quenching effect of different concentrations of acetic acid on the original signal and the reactivation effect of sodium carbonate.

[0045] Figure 2 These are imaging solutions taken using the fMOST system after quenching tissue blocks with acetic acid of different concentrations in a sodium carbonate environment.

[0046] Figure 3 This is a comparison of FITC signal detection in chemical chromatography (top image) and blackening chemical chromatography (bottom image).

[0047] Figure 4 This is a comparison of the imaging effects of blackening chromatography (top image) and blackening chemical chromatography (bottom image). Detailed Implementation

[0048] FITC (Fluororescein Isothiocyanate) is the most classic and widely used green fluorescent labeling probe in the life sciences. Its core principle is the covalent coupling of isothiocyanate groups with biomolecules to achieve fluorescent visualization of the target analyte. The labeling mechanism involves nucleophilic addition of isothiocyanate to the primary amino group (-NH2) of biomolecules (proteins, antibodies, peptides, etc.) under weakly alkaline conditions (pH 8.0-9.0), forming a stable thiourea bond and achieving covalent labeling. Its advantages include a mild reaction, high efficiency, stable conjugates, and minimal impact on the activity of biomolecules. Therefore, it is widely used in resin embedding of biological samples and fMOST imaging.

[0049] Example

[0050] The following specific examples illustrate the implementation of the present invention. Those skilled in the art can easily understand other advantages and effects of the present invention from the content disclosed in this specification. The present invention can also be implemented or applied through other different specific embodiments, and various details in this specification can also be modified or changed based on different viewpoints and applications without departing from the spirit of the present invention.

[0051] Before further describing specific embodiments of the present invention, it should be understood that the scope of protection of the present invention is not limited to the specific embodiments described below; it should also be understood that the terminology used in the embodiments of the present invention is for describing specific embodiments and not for limiting the scope of protection of the present invention.

[0052] When numerical ranges are given in the embodiments, it should be understood that, unless otherwise stated in the invention, both endpoints of each numerical range and any value in between may be selected. Unless otherwise stated, all percentages mentioned in this invention are mass percentages. Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention pertains.

[0053] Where specific techniques or conditions are not specified in the examples, they shall be performed in accordance with the techniques or conditions described in the literature in this field, or in accordance with the product instructions. Reagents or instruments whose manufacturers are not specified are all conventional products that can be purchased through legitimate channels.

[0054] The biological tissue samples, reagents, and equipment used in the following examples and comparative examples are as follows:

[0055] 1. Preparation of experimental materials and reagents

[0056] 1.1 Biological tissue samples: Healthy adult C57BL / 6 mice were selected, and after sacrifice, the whole brain of the mice was quickly separated and trimmed into tissue blocks with a volume of approximately 5mm×5mm×5mm for later use;

[0057] 1.2 Reagents: 4% paraformaldehyde (PFA) solution, PBS buffer (pH 7.4), 95% ethanol, 100% ethanol, Sudan Black B (SBB), ABVN (azobisisobutyronitrile), HM20 resin, acetic acid (HAc), gelatin capsules, sodium carbonate, PI staining solution (propidium iodide staining solution, optional, used for real-time staining to obtain tissue architecture information);

[0058] 1.3 Instruments: constant temperature and pressure drying oven, fMOST imaging system, centrifuge, and ultra-clean workbench.

[0059] Example 1: A method for embedding surface fluorescence chromatography resin in immunostained samples and fMOST imaging

[0060] A method for embedding surface fluorescent chromatography resin in immunostained samples, comprising the following steps:

[0061] S1. Pretreatment of biological tissue samples: Mouse brains were fixed with 4% PFA solution and then rinsed multiple times with PBS buffer. Biological tissue samples were fluorescently labeled with FITC fluorescent probes and then rinsed multiple times with PBS buffer.

[0062] Additional notes: FITC fluorescent probe labeling is a routine pre-operation for fluorescence imaging of biological tissue samples. Labeling methods can include immunofluorescence labeling or in vivo labeling. This embodiment uses immunofluorescence labeling, specifically: the rinsed tissue block is placed in FITC-labeled secondary antibody solution and incubated overnight at 4°C. The next day, it is rinsed three times with PBS buffer for 15 minutes each time to complete the labeling. After labeling, it is necessary to ensure that the tissue sample carries the FITC fluorescent probe, but it is not necessary to maintain its activated state. Subsequent acidification can regulate it to a transient quenched state to prepare for subsequent imaging reactivation.

[0063] S2, Gradient Ethanol Dehydration and Tissue Blackening: The pretreated biological tissue samples were sequentially dehydrated in 50%, 75%, and 95-100% ethanol gradients for 2 hours each, and then transferred to SBB / EtOH solution for dehydration overnight at 4°C; the SBB / EtOH solution was obtained by adding 0.5% SBB to 100% ethanol and stirring to dissolve it.

[0064] In this step, gradient dehydration can avoid the drastic tissue shrinkage caused by the direct action of high concentrations of ethanol, and SBB pre-osmosis can initially establish a background suppression environment to reduce subsequent imaging interference.

[0065] S3. Gradient resin permeation: The dehydrated biological tissue sample is permeated sequentially with 50% resin / EtOH and 75% resin / EtOH for 2.5 hours each, then transferred to 100% resin for 2 hours. The solution is changed overnight, and the next day, fresh 100% resin is used for incubation for 36 hours. The resin is HM20 resin (the following steps in this embodiment also use this resin).

[0066] In this step, resin penetration is a slow physical diffusion process. If penetration is insufficient, it will lead to uneven monomer density between the center and the edge of the tissue, causing sample deformation or cavitation (bursting polymerization) after polymerization.

[0067] S4. Acidification treatment: The fully permeated biological tissue sample is transferred into an acidification resin solution and treated at 4°C for 13 hours; the acidification resin solution is obtained by adding 0.08% acetic acid to 100% resin mother liquor.

[0068] In this step, acetic acid can adjust the pH of the resin microenvironment. Since the FITC fluorescent probe labeled in step S1 is pH sensitive, the acidic environment can induce the fluorescent group to change from the naturally activated state to a temporary "quenched state". At this time, the FITC fluorescent signal is not activated and no obvious fluorescence can be detected, but the molecular structure of the fluorescent group is not destroyed and is in a reactivatable state, which lays the foundation for fluorescence reactivation during subsequent imaging. If the acidification time is insufficient, the acidic components cannot completely diffuse into the deep layer of the sample, and the fluorescence signal is difficult to reactivate effectively, resulting in low imaging brightness. If the acidification time is too long, the local pH will be too low, which will destroy the molecular structure of the fluorescent group and cause irreversible fluorescence quenching.

[0069] S5. Resin polymerization molding: The acidified biological tissue sample is placed in a gelatin capsule, SBB / ABVN / resin solution is added, and thermal polymerization is carried out to complete the embedding of the biological tissue sample.

[0070] The SBB / ABVN / resin solution was obtained by adding 0.5% SBB and 0.6% ABVN to the resin and stirring until homogeneous. The thermal polymerization was carried out in a sealed constant temperature and pressure oven under the following conditions: 38°C for 4 hours and 40°C for 8 hours.

[0071] In this step, during the polymerization process, ABVN acts as an initiator to induce the resin monomers to undergo a cross-linking reaction, solidifying the tissue sample into a rigid embedding block. This embedding block can be directly used for section imaging in the fMOST system.

[0072] S6, fMOST imaging

[0073] The polymerized rigid embedded block was removed from the gelatin capsule and imaged in a sodium carbonate solution environment of the fMOST system with a concentration of 5.8 g / L.

[0074] In this step, sodium carbonate solution serves as the imaging environment, which can neutralize the acidification effect of the embedding resin containing acetic acid, thereby reactivating the FITC signal. During the imaging process, an appropriate amount of PI staining solution can be added to the imaging environment for real-time staining according to experimental requirements, in order to obtain the tissue sample's construction information.

[0075] Example 2: A method for embedding surface fluorescence chromatography resin in immunostained samples and fMOST imaging

[0076] A method for embedding surface fluorescent chromatography resin in immunostained samples, comprising the following steps:

[0077] S1. Pretreatment of biological tissue samples: Mouse brains were fixed with 4% PFA solution and then rinsed multiple times with PBS buffer. Biological tissue samples were fluorescently labeled with FITC fluorescent probes (same as in Example 1) and then rinsed multiple times with PBS buffer.

[0078] S2, Gradient ethanol dehydration and tissue blackening: The pretreated biological tissue samples were dehydrated sequentially by 50%, 75%, and 95-100% ethanol gradients for 1 hour each, and then transferred to SBB / EtOH solution for dehydration overnight at 4°C.

[0079] The SBB / EtOH solution was obtained by adding 0.6% SBB to 95% ethanol and then stirring to dissolve it.

[0080] S3. Gradient resin permeation: The dehydrated biological tissue sample is permeated sequentially with 50% resin / EtOH and 75% resin / EtOH for 3 hours each, then transferred to 100% resin for 1 hour, the solution is changed overnight, and the next day it is replaced with fresh 100% resin and incubated for 48 hours; the resin is GMA resin (the following steps in this embodiment also use this resin).

[0081] S4. Acidification treatment: The fully permeated biological tissue sample is transferred into an acidification resin solution and treated at 4°C for 15 hours; the acidification resin solution is obtained by adding 0.1% acetic acid to 100% resin mother liquor.

[0082] S5. Resin polymerization molding: The acidified biological tissue sample is placed in a gelatin capsule, SBB / ABVN / resin solution is added, and thermal polymerization is carried out to complete the embedding of the biological tissue sample.

[0083] The SBB / ABVN / resin solution was obtained by adding 0.6% SBB and 0.8% ABVN to the resin and stirring until homogeneous; the thermal polymerization was carried out in a sealed constant temperature and pressure oven at a temperature of 50°C for 6 hours.

[0084] S6, fMOST imaging

[0085] The polymerized rigid embedding block was removed from the gelatin capsule and imaged in a sodium carbonate solution (6 g / L) environment within the fMOST system. During imaging, an appropriate amount of PI staining solution could be added to the imaging environment for real-time staining, as needed, to obtain tissue sample construction information.

[0086] Example 3: A method for embedding surface fluorescence chromatography resin in immunostained samples and fMOST imaging

[0087] A method for embedding surface fluorescent chromatography resin in immunostained samples, comprising the following steps:

[0088] S1. Pretreatment of biological tissue samples: Mouse brains were fixed with 4% PFA solution and then rinsed multiple times with PBS buffer. Biological tissue samples were fluorescently labeled with FITC fluorescent probes (same as in Example 1) and then rinsed multiple times with PBS buffer.

[0089] S2, Gradient ethanol dehydration and tissue blackening: The pretreated biological tissue samples were dehydrated sequentially by 50%, 75%, and 95-100% ethanol gradients for 1 hour each, and then transferred to SBB / EtOH solution for dehydration overnight at 4°C.

[0090] The SBB / EtOH solution was obtained by adding 0.6% SBB to 100% ethanol and then stirring to dissolve it.

[0091] S3. Gradient resin permeation: The dehydrated biological tissue sample is permeated sequentially with 50% resin / EtOH and 75% resin / EtOH for 2 hours each, then transferred to 100% resin for 3 hours. The solution is changed overnight, and the next day, fresh 100% resin is used for incubation for 24 hours. The resin is BMA resin (the following steps in this embodiment also use this resin).

[0092] S4. Acidification treatment: The fully permeated biological tissue sample is transferred into an acidification resin solution and treated at 4°C for 12 hours; the acidification resin solution is obtained by adding 0.05% acetic acid to 100% resin mother liquor.

[0093] S5. Resin polymerization molding: The acidified biological tissue sample is placed in a gelatin capsule, SBB / ABVN / resin solution is added, and thermal polymerization is carried out to complete the embedding of the biological tissue sample.

[0094] The SBB / ABVN / resin solution was obtained by adding 0.5% SBB and 0.45% ABVN to the resin and stirring until homogeneous; the thermal polymerization was carried out in a sealed constant temperature and pressure oven at a temperature of 38°C for 24 hours.

[0095] S6, fMOST imaging

[0096] The polymerized rigid embedding block was removed from the gelatin capsule and imaged in a sodium carbonate solution (5.5 g / L) environment using the fMOST system. During imaging, an appropriate amount of PI staining solution could be added to the imaging environment for real-time staining, as needed, to obtain tissue composition information.

[0097] Comparative Example 1: A chemical chromatography embedding method and fMOST imaging

[0098] A chemical chromatography embedding method includes the following steps:

[0099] S1. Pretreatment of biological tissue samples: Same as in Example 1.

[0100] S2, Gradient Ethanol Dehydration: The pretreated biological tissue samples were sequentially dehydrated in 50%, 75%, and 95-100% ethanol gradients for 2 hours each, and then placed in a 95-100% ethanol solution for overnight dehydration. The solution was kept at 4°C throughout the process.

[0101] S3. Gradient resin permeation: The dehydrated biological tissue sample was permeated sequentially with 50% resin / EtOH and 75% resin / EtOH for 2.5 hours each, then transferred to 100% resin for 2 hours. The solution was changed overnight, and the sample was incubated with fresh 100% resin for 36 hours the next day. The resin used was BMA resin (the following steps in this comparative example also use this resin).

[0102] S4. Acidification treatment: The fully permeated biological tissue sample is transferred into an acidification resin solution and treated at 4°C for 13 hours; the acidification resin solution is obtained by adding 0.08% acetic acid to 100% resin mother liquor.

[0103] S5. Resin polymerization molding: The acidified biological tissue sample is placed in a gelatin capsule, ABVN / resin solution is added, and thermal polymerization is carried out to complete the embedding of the biological tissue sample.

[0104] The ABVN / resin solution was obtained by adding 0.3% ABVN to the resin and stirring until homogeneous; the thermal polymerization was carried out in a sealed constant temperature and pressure oven under the following conditions: 38℃ for 4 hours and 40℃ for 8 hours.

[0105] S6, fMOST imaging

[0106] The polymerized rigid embedding block was removed from the gelatin capsule and imaged in a sodium carbonate solution (5.8 g / L) environment within the fMOST system. During imaging, an appropriate amount of PI staining solution could be added to the imaging environment for real-time staining, as needed, to obtain tissue composition information.

[0107] Comparative Example 2: A blackening tomography embedding method and fMOST imaging

[0108] A chemical chromatography embedding method includes the following steps:

[0109] S1. Pretreatment of biological tissue samples: Same as in Example 1.

[0110] S2, Gradient Ethanol Dehydration and Tissue Blackening: The pretreated biological tissue samples were sequentially dehydrated in 50%, 75%, and 95-100% ethanol gradients for 2 hours each, and then transferred to SBB / EtOH solution for dehydration overnight at 4°C; the SBB / EtOH solution was obtained by adding 0.5% SBB to 100% ethanol and stirring to dissolve it.

[0111] S3. Gradient resin permeation: The dehydrated biological tissue sample was permeated sequentially with 50% resin / EtOH and 75% resin / EtOH for 2.5 hours each, then transferred to 100% resin for 2 hours. The solution was changed overnight, and the sample was incubated with fresh 100% resin for 36 hours the next day. The resin used was BMA resin (the following steps in this comparative example also use this resin). In this step, the resin / EtOH solution contained 0.6% SBB.

[0112] S4. Acidification treatment: The fully permeated biological tissue sample is transferred into an acidification resin solution and treated at 4°C for 12 hours; the acidification resin solution is obtained by adding 0.08% acetic acid to 100% resin mother liquor.

[0113] S5. Resin polymerization molding: The acidified biological tissue sample is placed in a gelatin capsule, SBB / ABVN / resin solution is added, and thermal polymerization is carried out to complete the embedding of the biological tissue sample.

[0114] The SBB / ABVN / resin solution was obtained by adding 0.5% SBB and 0.6% ABVN to the resin and stirring until homogeneous. The thermal polymerization was carried out in a sealed constant temperature and pressure oven under the following conditions: 38°C for 4 hours and 40°C for 8 hours.

[0115] S6, fMOST imaging

[0116] The polymerized rigid embedding block was removed from the gelatin capsule and imaged in a sodium carbonate solution (5.8 g / L) environment within the fMOST system. During imaging, an appropriate amount of PI staining solution could be added to the imaging environment for real-time staining, as needed, to obtain tissue composition information.

[0117] The fMOST imaging results of Examples 1-3 showed that the tissue embedding blocks were homogeneous, free of bubbles and cracks, and the slicing process was smooth. During the experiment, the SBB background inhibitor and chemical chromatography interacted synergistically. By introducing HAc to selectively quench the FITC signal outside the focal plane, the layer-by-layer signal resolution effect of optical chromatography was simulated at the chemical level, effectively suppressing the interference of out-of-focus signals on the current imaging layer, allowing the FITC signal of the target layer to be clearly highlighted. Combined with the background suppression effect of SBB, the imaging background interference was significantly reduced, resulting in stable and bright FITC fluorescence signals, clear imaging, and clear observation of the fine structures inside the tissue. If PI staining solution is added, its specific binding with cellular DNA can display the cell distribution, tissue boundaries, and other structural information of the tissue in real time, further assisting in the interpretation of FITC signal localization. If PI staining solution is omitted, FITC signal imaging alone can still meet the core requirements of high-resolution imaging of the fMOST system. However, the fMOST imaging of FITC signals in Comparative Example 1 was significantly less clear than the imaging in Examples 1-3.

[0118] Figure 1 To capture fluorescence signals before and after quenching and recovery using confocal imaging and to statistically compare cell brightness, the quenching effect of different concentrations of acetic acid (adjusted based on Example 1) on the original signal was compared with that of sodium carbonate reactivation. The quenching ratio of acetic acid on the original signal and the recovery ratio of sodium carbonate on the quenched signal were quantitatively calculated. The figure illustrates that when the acetic acid concentration is less than 5%, the signal quenched by acetic acid can be recovered by sodium carbonate.

[0119] Figure 2-4 The fluorescence was collected using the fMOST system after quenching with acetic acid and recovery with sodium carbonate.

[0120] in Figure 2 The imaging results of the sodium carbonate environment after quenching tissue blocks with 0.1%, 0.5%, and 1% acetic acid (adjusting the acetic acid concentration based on Example 1) were obtained using the fMOST system. It is shown that the signals outside the tissue blocks were completely quenched and could not be recovered due to the high concentration of 0.5% and 1% acetic acid. At the same time, the high concentration of acetic acid brought higher tissue autofluorescence, resulting in very low signal quality. However, the 0.1% concentration of acetic acid showed good quenching and sodium carbonate recovery performance.

[0121] in Figure 3The image shows a comparison of FITC signal detection between chemical chromatography (top image, embedding method of Comparative Example 1) and blackening chemical chromatography (bottom image, embedding method of Example 1). The results show that after adding SBB background inhibitor and superimposing chemical chromatography, the FITC signal is stable and bright.

[0122] in Figure 4 The image shows a comparison of the imaging effects of blackening chromatography (top image, the embedding method of Comparative Example 2) and blackening chemical chromatography (bottom image, the embedding method of Example 1). The results show that after adding the SBB background inhibitor and superimposing chemical chromatography, the halo in the neuronal cell body region is low and the imaging chromatography effect is good.

[0123] The above embodiments and comparative examples illustrate that the embedding method used in this invention for embedding biological tissues significantly reduces imaging background interference, resulting in stable and bright FITC fluorescence signals, clear imaging, and clear observation of the fine structures inside the tissue.

[0124] Although the present invention has been described in detail through the preferred embodiments above, it should be understood that the above description should not be considered as a limitation of the present invention. Various modifications and substitutions to the present invention will be apparent to those skilled in the art after reading the above description. Therefore, the scope of protection of the present invention should be defined by the appended claims.

Claims

1. A method for embedding surface fluorescent chromatography resin in immunostained samples, characterized in that, The method includes the following steps: S1. Pretreatment of biological tissue samples: Fix and label biological tissue samples with fluorescent probes for later use; S2, Gradient ethanol dehydration and tissue blackening: The pretreated biological tissue samples were dehydrated by a gradient of different concentrations of ethanol, and then transferred to SBB / EtOH solution for dehydration overnight at 4°C. S3, Gradient Resin Permeation: The dehydrated biological tissue sample is permeated with a gradient of resin / ethanol at different concentrations, then transferred to 100% resin for permeation, the solution is changed and left overnight, and then the 100% resin is replaced and incubated the next day. S4. Acidification treatment: Transfer the fully permeated biological tissue sample into the acidification resin solution and treat at 4°C. S5. Resin Polymerization Molding: The acidified biological tissue sample is placed in a gelatin capsule, and SBB / ABVN / resin solution is added. Thermal polymerization is then carried out to complete the embedding of the biological tissue sample.

2. The method as described in claim 1, characterized in that, In step S1, The biological tissue sample is mouse brain or mouse peripheral organ tissue; Biological tissue samples were fixed with 4% PFA solution and then rinsed multiple times with PBS buffer. Biological tissue samples were fluorescently labeled using FITC fluorescent probes and then rinsed multiple times with PBS buffer.

3. The method as described in claim 1, characterized in that, In step S2, The pretreated biological tissue samples were sequentially dehydrated using 50%, 75%, and 95-100% ethanol gradients. And / or dehydrate for 1-3 hours at different concentrations of ethanol gradient; And / or the SBB / EtOH solution is obtained by adding 0.5-0.6% SBB to 95-100% ethanol and then stirring to dissolve it.

4. The method as described in claim 1, characterized in that, In step S3, Biological tissue samples were permeated sequentially with 50% resin / EtOH and 75% resin / EtOH. And / or allow to permeate for 2-3 hours at different concentrations of resin / EtOH gradients; And / or transfer the gradient-permeable biological tissue sample into 100% resin for 1-3 hours; And / or incubate in a fresh 100% resin solution for 24-48 hours the following day; And / or the resin is an acrylic resin, such as HM20 resin, GMA resin or BMA resin.

5. The method as described in claim 1, characterized in that, In step S4, The acidified resin solution is obtained by adding 0.05-0.1% acetic acid to 100% resin mother liquor; And / or treat biological tissue samples in an acidified resin solution for 12-15 hours; And / or the resin is an acrylic resin, such as HM20 resin, GMA resin or BMA resin.

6. The method as described in claim 1, characterized in that, In step S5, The SBB / ABVN / resin solution is obtained by adding 0.5-0.6% SBB and 0.45-0.8% ABVN to the resin and stirring until homogeneous. And / or the resin is an acrylic resin, such as HM20 resin, GMA resin or BMA resin; And / or the thermal polymerization is carried out in a closed, constant temperature and pressure oven; And / or the temperature of the thermal polymerization is 38-50°C, and the time of the thermal polymerization is 6-24 hours; And / or the thermal polymerization conditions are 38°C for 4 hours or 40°C for 8 hours.

7. An imaging method for biological tissue samples, characterized in that, The method includes the step of imaging a biological tissue sample obtained by the embedding method according to any one of claims 1-6 in a sodium carbonate solution environment of the fMOST system.

8. The method as described in claim 7, characterized in that, The concentration of the sodium carbonate solution is 5.5-6 g / L.

9. The method as described in claim 7, characterized in that, During the imaging process, PI real-time staining is combined to obtain the structural information of biological tissue samples.

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