Methods for mounting biological samples on surfaces

Abstract

The technology provided herein relates to methods of mounting / attaching a biological sample onto a surface by contacting the biological sample on the surface with a reducing agent.

Description

[Technical field]

[0001] Field of Disclosure The technology provided herein relates to methods of mounting / adhering a biological sample onto a surface by contacting the biological sample on the surface with a reducing agent. [Background technology]

[0002] background Tissue sections, cell tissues or other body fluids or biological materials obtained from clinical specimens or animal experiments are often mounted, fixed and preserved in a form suitable for microscopic examination. Mounting is the process of attaching the specimen to a microscope slide and protecting it from physical damage. Generally, in microscopy, a slide of the material or specimen to be studied is prepared by placing said sample or material on a (bottom) slide and then covering said sample with a (top) cover. Generally, in light microscopy or similar techniques, slides and covers of glass or another suitable transparent material are used.

[0003] However, some tissue section samples adhere strongly to the microscope slide and survive several incubation and reaction steps, while other samples (e.g., lung) may be lost during the staining process.

[0004] A typical mounting process uses coated slide surfaces to mount tissue sections and enhance their adhesion to microscope slides. As mentioned above, typical surface modifications are based on polylysine, collagen, gelatin, fibronectin, laminin, vitronectin, PEG, and materials that enhance the adhesion of tissue sections to the surface of microscope slides. Other methods claim modification of the glass surface by plasma treatment of the glass, or deposition of inorganic materials such as silanes or titanium dioxide (WO 2017 / 158238 A1). A drawback of coatings is their limited half-life, often of only a few months.

[0005] Therefore, some researchers also use tissue treatments such as incubation with formaldehyde to increase the adhesion of tissue sections to glass slides. It is also well known that tissue sections from collected paraffin-embedded tissue blocks can change their adhesion to surfaces. Formaldehyde, which is also used in paraffin-embedded tissue samples for stabilization, results in non-directional cross-linking of biomolecules that can interfere with downstream analysis.

[0006] Downstream analysis of small amounts of analytes in biological and non-biological samples has become routine in clinical and analytical environments. Numerous analytical methods have been established for this purpose. Some of these use coding techniques that assign a specific readable code to a specific first analyte that differs from the code assigned to a specific second analyte.

[0007] Against this background, the underlying subject of the present disclosure is to provide a method for mounting biological samples on a surface by means that can reduce or even avoid the drawbacks of the prior art methods. Summary of the Invention [Problem to be solved by the invention]

[0008] The present disclosure relates to new methods and compositions for mounting (or adhering / fixing) biological samples onto a surface such as a microscope slide. [Means for solving the problem]

[0009] For example, microscopy is a widely used method in biology and pathology. Some tissue section samples adhere strongly to microscope slides and survive several incubation and reaction steps. Other samples (e.g., lung) may be lost during the staining procedure. The disclosed method enhances the success of the analysis of these samples, which are quite frequently lost during the process. Effect of the Invention

[0010] In a first aspect, an embodiment of the present disclosure provides a method of mounting a biological sample on a surface, comprising: i) applying a biological sample to a surface; ii) contacting the biological sample on the surface with a reducing agent, optionally followed by iii) contacting the biological sample on the surface with an oxidizing agent. The present invention relates to a method comprising the steps of:

[0011] In a second aspect, an embodiment of the present disclosure provides a method for mounting (or adhering / fixing) tissue on a slide and for microscopy, comprising: i) applying the tissue to the surface of a slide; ii) contacting the tissue on the slide with a solution of a reducing agent; iii) drying the slides to remove excess solvent; iv) contacting the tissue on the slide with an aqueous solution of an oxidizing agent. and microscopic examination of said tissue on said slide.

[0012] In a third aspect, an embodiment of the present disclosure provides a kit for mounting a biological sample on a surface, comprising: a) a reducing agent, and b) Oxidizing agent The present invention relates to a kit comprising:

[0013] Before describing the present disclosure in detail, it should be understood that the present disclosure is not limited to the specific component parts of the method steps described. It should also be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting. It should be noted that as used in this specification and the appended claims, the singular forms "a," "an," and "the" include singular and / or plural referents unless the context clearly dictates otherwise. Furthermore, when a parameter range bounded by numerical values ​​is given, it should be understood that the range is deemed to include these limiting values. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0014] Detailed Description of the Disclosure Disclosed herein are new methods and kits for mounting (or adhering) biological samples onto surfaces such as microscope slides. The disclosure particularly describes the use of a reducing agent in combination with an oxidizing agent to enhance the success of mounting and subsequent analysis of biological samples that are very frequently lost during analytical processes such as staining processes for microscopy.

[0015] In some embodiments, the present disclosure provides: 1) Contacting a biological sample with a surface 2) incubating the biological sample with at least a reducing agent; 3) Perhaps then, at least, the reduction reaction is quenched with an oxidizing agent. The present invention describes the processing of a biomolecule-containing sample by

[0016] Before step 1, the biomolecule-containing sample may be incubated with enzymatic, chemical, or physical treatment, or a combination thereof, to change the characteristics of the sample. Before step 1, the tissue sample may be cut by microtome or cryotome, and the tissue slice may be used for step 1. Before step 1, cells, organoids, organic cultures, or other biological materials may be incubated in medium or solution.

[0017] Step 1 may be followed by further treatments such as deparaffinization, one or more enzymatic treatments, one or more chemical treatments, one or more physical treatments, or a mixture thereof. Steps 2 and 3 reduce the risk of loosening the contact of the biological sample to the surface. Only step 2 may be performed. The reduction reaction can reduce disulfide bridges in the biological molecule-containing sample. The reaction that stops the reduction reaction can oxidize the reduced thiol groups. The biological molecule-containing sample is immobilized on a flat surface during processing for further analysis, such as microscopy, spectroscopy, or other analysis.

[0018] Surprisingly, the inventors have found that, for example, a reaction that reduces disulfide bonds in a first step and oxidizes thiol groups in a second reaction results in better adhesion of biomolecule-containing materials to surfaces such as glass or plastic.

[0019] As discussed above, an embodiment of the present disclosure is a method for mounting a biological sample on a surface, comprising: i) applying a biological sample to a surface; ii) contacting said biological sample on said surface with a reducing agent, optionally followed by iii) contacting the biological sample on the surface with an oxidizing agent, optionally followed by iv) staining / analysing the mounted biological sample on said surface. The present invention relates to a method comprising the steps of:

[0020] Further embodiments of the present disclosure include a method of mounting a biological sample on a surface, comprising: i) applying a biological sample to a surface; ii) contacting the biological sample on the surface with a reducing agent; and then iii) contacting the biological sample on the surface with an oxidizing agent, optionally followed by iv) staining / analysing the mounted biological sample on said surface. The present invention relates to a method comprising the steps of:

[0021] "Biological sample" means any material including, but not limited to, blood, serum, body fluids and tissue biopsy samples collected from a test subject, and any tangible material derived directly or indirectly therefrom. "Biological sample" or "biological specimen" is used herein to refer to, for example, a tissue section or a cytochemical smear. Prior to mounting, the biological sample / specimen may be subjected to a series of physical and chemical manipulations, including sectioning and staining. Such physical and chemical manipulations are known to those skilled in the art and therefore will not be described, but will be described very briefly herein below. Once subjected to the aforementioned physical and chemical manipulations, the biological sample / specimen may be referred to as a "histochemical section" or a "cytochemical smear". Thus, a "histochemical section" refers to a solid sample of biological tissue that has been frozen or chemically fixed and / or hardened by embedding in wax or plastic, sliced ​​into thin sheets, typically a few microns thick, and adhered onto a surface such as a microscope slide. Furthermore, a "cytochemical smear" refers to a suspension of cells, such as blood cells, to be fixed and adhered to a surface such as a microscope slide.

[0022] In particular, a biological sample is a biomolecule-containing sample that can be described by its content of biomolecules carrying at least oxidized disulfide bridges. The sample may be dead or alive. It is not necessary that all biomolecules in the sample contain oxidized thiol groups. A biomolecule-containing sample can be a biomolecule, a cell (bacteria, archaea, eukaryotes, etc.), a tissue sample, a tissue section, a whole organ sample, a whole organism, or an extract of multiple organisms. A sample may also contain other materials that do not contain biomolecules. A sample also includes a sample to which an extract of a biomolecule has been added.

[0023] A "biomolecule" is a molecule that can be found in an organism and / or cell. It also includes molecules that have been manipulated, digested, or modified by any type of biological, enzymatic, chemical, or physical process. It also includes extracted biomolecules that are added to a sample, such as a biological sample.

[0024] Thus, in some advantageous embodiments, biological sample comprises biomolecule, cell, cell culture, tissue section, organoid and / or organic culture, organ, or whole organism.In particular, biological sample comprises biomolecule that contains at least disulfide bridge, especially oxidized thiol group.

[0025] In some advantageous embodiments, the biological sample comprises a biomolecule, a cell such as a prokaryotic cell, an archaeal cell or a eukaryotic cell, a tissue sample, a tissue section, a whole organ sample, a whole organism, or an extract of multiple organisms. In particular, the biological sample comprises blood, serum, body fluids or tissue, such as a biopsy sample.

[0026] In some embodiments, the biological sample is a tissue sample, and the tissue is cut into tissue sections by a microtome or cryotome before being applied to the surface. In particular, the biological sample includes lung or skin tissue. In some embodiments, the biological sample is incubated in a medium or solution before being applied to the surface.

[0027] In some further advantageous embodiments, the biological sample is treated by enzymatic, chemical or physical treatment or a combination thereof before being applied to the surface. In an embodiment of the present disclosure, the biological sample comprises a formalin-fixed paraffin-embedded (FFPE) tissue. In particular, in an embodiment of the method according to the present disclosure, the formalin-fixed paraffin-embedded (FFPE) tissue is deparaffinized after being applied to the surface, in particular by one or more enzymatic treatments, one or more chemical treatments, one or more physical treatments, or a mixture thereof.

[0028] In some advantageous embodiments, the biological sample / specimen is fixed by a method according to the present disclosure prior to mounting a coverslip over the biological sample / specimen on a microscope slide.

[0029] As used herein, "fixing," "adhering" or "mounting a biological sample / specimen" on a surface such as a microscope slide refers to a sample of biological cells, such as biological tissue, that has been chemically treated to stabilize proteins and strengthen cellular structures, particularly membranes, against destruction by solvent changes, temperature changes, mechanical stress, and drying. The cells may be fixed in suspension or contained in a sample of tissue that may be obtained during dissection, biopsy, or surgery.

[0030] Additionally, the cells or tissues to be examined may be embedded in warm liquid paraffin wax. The wax both surrounds and permeates the tissue and hardens upon cooling, thereby supporting the tissue externally and internally. The resulting solid paraffin block is then trimmed to the appropriate shape before sectioning. If ultra-thin sections are required, the use of harder embedding and permeating materials, such as epoxy plastics, may be required. Such materials, initially in liquid form, are poured into small molds containing the fixed tissue pieces, and upon heating, the liquid polymerizes to form a hard plastic.

[0031] The trimmed block containing the embedded sample is sectioned using a microtome, an instrument in which the block moves sequentially over a knife blade that cuts the block into a series of thin sections. Such sections are then mounted, i.e., deposited or glued, onto microscope slides and stained with various colored dyes or chromogens that specifically adhere to different molecular components of the cells. At this point, a coverslip can be placed over the fixed tissue or cell specimen.

[0032] As used herein, "coverslip" refers to a thin slip of glass, plastic, or other transparent polymeric material used to cover a biological specimen on a microscope slide to be viewed under a microscope. The cover slip must be long and wide enough to cover the entire biological sample / specimen. As used herein, "placing a cover slip" refers to placing a cover slip over a microscope slide having a biological sample / specimen thereon.

[0033] The surface on which the biological sample is mounted / fixed / attached may be flat or rounded, smooth or rough, hydrophilic or oleophilic. Furthermore, the surface may be made of various materials and may be based on glass, plastic, metal, quartz or any other type of material from which a surface can be made. In an advantageous embodiment, the surface is on or is a microscope slide. Furthermore, the surface may be modified or coated to introduce further functionalities.

[0034] A "reducing agent" or reductant loses electrons and becomes oxidized in a chemical reaction. A reducing agent is typically in one of its lower possible oxidation states and is known as an electron donor. A reducing agent becomes oxidized by losing an electron in an oxidation-reduction reaction. Examples of reducing agents include earth metals, formic acid, and sulfite compounds. As used herein, "reducing agent" includes all agents and conditions that can specifically reduce disulfide bonds. The reducing ability of a reducing agent can be measured as the electrode potential compared to the potential of a standard electrode. Reducing agents can change their reducing ability depending on the specific reaction conditions, such as changing temperature, pH, concentration, or other parameters (see Pettrucci, Ralph H. General Chemistry: Principles and Modern Applications, 9th ed. Upper Saddle River: Pearson Prentice Hall, 2007; Oxtoby, David W., HP Gillis, and Alan Campion. Principles of Modern Chemistry, 6th ed. Belmont: Thomson Brooks / Cole, 2008). Thus, one skilled in the art can vary the agents and reaction parameters to reduce disulfide bonds in biomolecules. Common reducing agents used in biochemical reactions are beta-mercaptoethanol, dithiothreitol (DTT), or dithioerythrit. In addition, many metal-based compounds can be used as reducing agents, such as sodium borohydride (NaBH4) or lithium aluminum hydride (LiAlH4). To prequalify a reducing agent for the purposes described herein, one of skill in the art can select a reagent from a galvanic series.

[0035] In some advantageous embodiments, the reducing agent used in the methods according to the present disclosure is selected from the group consisting of beta-mercaptoethanol, dithiothreitol (DTT), dithioerythritol, sodium borohydride (NaBH4), lithium aluminum hydride (LiAlH4), or another reducing agent of the galvanic series.

[0036] An "oxidizing agent" or oxidant is one that gains electrons and is reduced in a chemical reaction. Also known as an electron acceptor, an oxidizing agent is usually in one of its higher possible oxidation states in order to gain electrons and be reduced. As used herein, "oxidizing agent" includes all agents and conditions that can oxidize thiol groups, particularly halogens, potassium nitrate, and nitric acid. For example, the oxidizing ability of an oxidizing agent can be measured as an electrode potential compared to the potential of a standard electrode. Oxidizing agents can change their oxidizing capabilities by changing the specific reaction conditions, such as temperature, pH, concentration, or other parameters (see Pettrucci, Ralph H. General Chemistry: Principles and Modern Applications, 9th ed. Upper Saddle River: Pearson Prentice Hall, 2007; Oxtoby, David W., HP Gillis, and Alan Campion. Principles of Modern Chemistry, 6th ed. Belmont: Thomson Brooks / Cole, 2008). In this regard, those skilled in the art can vary the agents and reaction parameters to oxidize thiol groups in biomolecules. In particular, biological samples / specimens, particularly biomolecule-containing samples, are treated with reducing and oxidizing reagents to make the samples adhere more firmly to the surface. Common oxidizing agents used in biochemical reactions are peroxides such as hydrogen peroxide (HO), organic peroxides (e.g., tert-butyl hydroperoxide), or other oxidizing reagents that can be selected by the researcher from sodium perborate, potassium permanganate, hypochlorite, and the galvanic series.

[0037] In some advantageous embodiments, the oxidizing agent used in the methods according to the present disclosure is selected from the group consisting of hydrogen peroxide (HO), an organic peroxide such as tert-butyl hydroperoxide, sodium perborate, potassium permanganate, hypochlorite, or another oxidizing reagent of the galvanic series.

[0038] As mentioned above, the present disclosure provides a method for mounting / adhering / fixing tissue on a slide and for microscopy, comprising: -Applying tissue to the surface of the slide; - contacting the tissue on the slide with a solution of a reducing agent; - drying the slides to remove excess solvent; - contacting the tissue on the slide with an aqueous solution of an oxidizing agent; - microscopic examination of the tissue on the slide The present invention also relates to a method and a microscopy comprising:

[0039] Additionally, the present disclosure also relates to a kit for mounting / adhering / fixing a biological sample on a surface comprising the above-mentioned reducing and oxidizing agents.

[0040] As mentioned above, the biological sample mounted on the surface may be stained and / or analyzed. Staining is used to highlight important features of the biological sample, such as tissue, as well as to enhance the contrast of the tissue, for example. Hematoxylin is a basic dye commonly used in this process, staining the nucleus and giving it a bluish color, while eosin (another staining dye used in histology) stains the nucleus of the cell and gives it a pinkish stain. However, there are several other staining techniques used for specific cells and components (Black, 2012). Staining is a medical process commonly used in the medical diagnosis of tumors, where dye colors are applied to the posterior and anterior borders of the sample tissue to locate diseased or tumor cells or other pathological cells (Musumeci, 2014). In biological research, staining is used to label cells, mark nucleic acids, proteins, or for gel electrophoresis to assist microscopic examination (Jackson & Blythe, 2013). In some cases, various multiple staining techniques such as banding, double staining or multiple staining are used (Iyiola & Avwioro, 2011).

[0041] In some advantageous embodiments, the mounted / fixed biological samples on the surface are used for spatial transcriptomics (or spatial omics). * The spatial omics is analyzed by spatial omics), which means any type of analysis in which data from the sample is derived spatially from an in situ sample of a tissue or whole organism. The in situ sample may be a section of an organ or an organism. The in situ sample may be unprocessed or preprocessed in a manner required to improve the results. Spatial omics may include the detection of small molecule compounds of tissues or cells, proteins, DNA and / or RNA. More preferentially, spatial omics is limited to proteins, DNA and / or RNA. More preferentially, spatial omics is limited to DNA and / or RNA. More preferentially, spatial omics is limited to smFISH, in situ sequencing or nucleic acid capture methods. Even more preferentially, spatial omics is limited to smFISH. Even more preferentially, spatial omics is limited to any type of sequential smFISH.

[0042] In particular, spatial transcriptomics detection includes multiplexing methods for detecting different analytes in a pathogen-containing sample by sequential signal coding of said analytes as described in WO 2020 / 254519 A1, PCT / EP 2021 / 066620 or PCT / EP 2021 / 066668.

[0043] A further method for analyzing / staining surface mounted biological samples according to the present disclosure may be the analysis and detection of small amounts of analytes in biological samples, which has become routine in clinical and analytical environments. For this purpose, numerous analytical methods have been established, such as FISH, in situ sequencing, methods involving biomolecular capture or spectroscopy methods (e.g. mass spectrometry). Some of these use coding techniques that assign a specific readable code to a first analyte that differs from the code assigned to a specific second analyte.

[0044] One of the prior techniques in this field is the so-called "single-molecule fluorescent in situ hybridization" (smFISH), which was developed essentially to detect mRNA molecules in a sample. In Lubeck et al. (2014), Single-cell in situ RNA profiling by sequential hybridization, Nat. Methods 11(4), p.360-361, the mRNA of interest is detected via a specific directly labeled probe set. After one round of hybridization and detection, a set of mRNA-specific probes is eluted from the mRNA, and the same set of probes with other (or the same) fluorescent labels is used in the next round of hybridization and imaging, generating a gene-specific color-coded scheme over several rounds. The technique requires several different tagged probe sets per transcript and these probe sets must be denatured after every detection round.

[0045] Further developments of this technology do not use directly labeled probe sets. Instead, the oligonucleotides of the probe set provide nucleic acid sequences that act as initiators for hybridization chain reaction (HCR), a technique that allows for amplification of the signal. See Shah et al. (2016), In situ transcription profiling of single cells reveals spatial organization of cells in the mouse hippocampus, Neuron 92(2), p.342-357.

[0046] Another technique, called "multiplexed error-robust fluorescent in situ hybridization" (merFISH), has been described by Chen et al. (2015), RNA imaging. Spatially resolved, highly multiplexed RNA profiling in single cells, Science 348(6233):aaa6090. There, the mRNA of interest is detected via specific probe sets that provide additional sequence elements for subsequent specific hybridization of fluorescently labeled oligonucleotides. Each probe set provides four different sequence elements from a total of 16 sequence elements. After hybridization of the specific probe sets to the mRNA of interest, a so-called readout hybridization is performed. In each readout hybridization, one of the 16 fluorescently labeled oligonucleotides that is complementary to one of the sequence elements is hybridized. All readout oligonucleotides use the same fluorescent color. After imaging, the fluorescent signal is destroyed by illumination and the next round of readout hybridization is performed without a denaturation step. As a result, a binary code is generated for each mRNA species. A unique signal signature of 4 signals in 16 rounds is generated using only a single hybridization round for binding of a specific probe set to the mRNA of interest, followed by 16 rounds of hybridization of a readout oligonucleotide labeled with a single fluorescent color.

[0047] Further developments of this technology improve throughput by using two different fluorescent colors and removing the signal via disulfide cleavage between the readout oligonucleotide and the fluorescent label and alternative hybridization buffers. See Moffitt et al. (2016) High-throughput single-cell gene-expression profiling with multiplexed error-robust fluorescence in situ hybridization, Proc. Natl. Acad. Sci. US A. 113(39), p. 11046-11051.

[0048] The technique, called "intron seqFISH", is described in Shah et al. (2018), Dynamics and spatial genomics of the nascent transcriptome by intron seqFISH, Cell 117(2), p.363-376. There, the mRNA of interest is detected via a specific probe set that provides an additional sequence element for the subsequent specific hybridization of a fluorescently labeled oligonucleotide. Each probe set provides one of 12 possible sequence elements (representing the 12 "pseudocolors" used) per color-coding round. Each color-coding round consists of four successive hybridizations. In each of these successive hybridizations, three readout probes, each labeled with a different fluorophore, hybridize to the corresponding elements of the mRNA-specific probe set. After imaging, the readout probes are removed by 55% formamide buffer, followed by the next hybridization. After five color-coding rounds using four successive hybridizations, the color code is completed.

[0049] EP 0 611 828 discloses the use of bridging elements to mobilize signal generating elements to probes that specifically bind to analytes. More specific statements describe the detection of nucleic acids via specific probes that mobilize bridging nucleic acid molecules. This bridging nucleic acid ultimately mobilizes signal generating nucleic acids. This document also describes the use of bridging elements with two or more binding sites for signal generating elements for signal amplification, such as branched DNA.

[0050] Player et al. (2001), Single-copy gene detection using branched DNA (bDNA) in situ hybridization, J. Histochem. Cytochem. 49(5), p. 603-611, describes a method in which the nucleic acid of interest is detected via a specific probe set that provides an additional sequence element. In a second step, a preamplification oligonucleotide is hybridized to this sequence element. This preamplification oligonucleotide contains multiple binding sites for the amplification oligonucleotides that are hybridized in a later step. These amplification oligonucleotides provide multiple sequence elements for the labeled oligonucleotides. In this way, a branched oligonucleotide tree is constructed that results in an amplification of the signal.

[0051] A further development of this method, mentioned as described by Wang et al. (2012), RNAscope: a novel in situ RNA analysis platform for formalin-fixed, paraffin-embedded tissues, J. Mol. Diagn. 14(1), p. 22-29, uses a different design of mRNA-specific probes. Here, two of the mRNA-specific oligonucleotides must hybridize in close proximity to provide a sequence that can recruit the pre-amplified oligonucleotides. This method increases the specificity of the method by reducing the number of false positive signals.

[0052] Choi et al. (2010), Programmable in situ amplification for multiplexed imaging of mRNA expression, Nat. Biotechnol. 28(11), p.1208-1212, disclose a method known as "HCR-Hybridization Chain Reaction". The mRNA of interest is detected via a specific probe set that provides an additional sequence element. The additional sequence element is an initiator sequence to start the hybridization chain reaction. Essentially, the hybridization chain reaction is based on metastable oligonucleotide hairpins, which self-assemble into a polymer after the first hairpin is opened via the initiator sequence.

[0053] An additional analytical method according to the present disclosure is probabilistic cell typing by in situ sequencing (pciSeq), an approach that leverages previous scRNA-seq classification to identify cell types using multiplexed in situ RNA detection (Qian, X., Harris, KD, Hauling, T. et al. Probabilistic cell typing enables fine mapping of closely related cell types in situ Nat Methods 17, 101-106 (2020); Lee, J., Daugharthy, E., Schheiman, J. et al. Fluorescent in situ sequencing (FISSEQ) of RNA for gene expression profiling in intact cells and tissues Nat Protoc 10, 442-458 (2015)).

[0054] Further developments of the technology use so-called split-initiator probes that must hybridize closely to form the initiator sequence for HCR, similar to the RNAscope technology, which reduces the number of false positive signals; see Choi et al. (2018), Third-generation in situ hybridization chain reaction: multiplexed, quantitative, sensitive, versatile, robust Development 145(12).

[0055] Mateo et al. (2019), Visualizing DNA folding and RNA in embryos at single-cell resolution, Nature 568, p. 49ff, disclose a method called Optical Reconstruction of Chromatin Structure (ORCA), which aims to make chromosome lines visible.

[0056] EP 2 992 115 B1 describes a method of sequential single molecule hybridization, providing a technique for detecting and / or quantifying nucleic acids in cells, tissues, organs or organisms via sequential barcoding.

[0057] Methods and Examples 1) Experiment: Strength of tissue fixation Sixteen sections of PAXgene (QIAGEN) fixed tissues (mouse skin, intestine, lung, and kidney) were placed on two glass slides. An adhesive slide (ibidi) was attached to the glass slides to form 16 chambers that could be individually addressed with different reaction conditions. All wells were treated with a rehydration process using 100% isopropanol, 95% ethanol, 70% ethanol, and equilibration buffer. Individual solutions were added to individual wells in the presence or absence of 10 mM DTT and incubated overnight. For details and results, see Table 1 below.

[0058] Table 1: Conditions and results [Table 1]

[0059] As shown in Table 1, by using DTT pretreatment, the tissue is much better fixed to the glass slide. Without DTT pretreatment, one of eight tissue sections is completely lost, three of eight are detached, and certain areas are not in focus due to wobbling during microscopy. Thus, four of eight samples are not completely fixed. With DTT pretreatment, none of the eight samples are lost, and only one of the samples is wobbling.

[0060] 2) Experiment: Fixing lung samples to slides (CyFaV74) Eight lung samples (PAX gene immobilized; Qiagen) were immobilized on glass slides. All samples were treated in the same way with respect to rehydration, hybridization, staining and washing steps. Part of the samples was pre-incubated with solutions containing various concentrations of DTT for 30 minutes at 37°C. After DTT incubation, the reaction was stopped with H2O2. For details and results, see Table 2 below.

[0061] Table 2: Conditions and results [Table 2]

[0062] As shown in Table 2, lung samples without treatment with DTT or combined treatment with DTT and H2O2 were lost. All other samples that received DTT or combined DTT / H2O2 treatment could be analyzed. Treatment with H2O2 did not fix the samples to the slides.

[0063] 3) Experiment: Fixation of lung samples in the presence / absence of DTT (CyFaV77) Eight lung samples (PAX gene fixed; Qiagen) were treated with a solution containing sucrose and various concentrations of DTT (see table below). The samples were then cut and fixed on glass slides. All samples were treated in the same way with respect to rehydration, hybridization, staining and washing steps. A portion of the samples was incubated with a solution containing various concentrations of DTT for 30 minutes at 37°C. After incubation, all samples were incubated with 1% H2O2. For details and results, see Table 3 below.

[0064] Table 3: Conditions and results [Table 3]

[0065] As shown in Table 3, lung samples without DTT treatment were lost. Other samples that received DTT or combined DTT / H2O2 treatment could be analyzed with higher probability.

[0066] 4) Experiment: Is the sensitivity of smFISH experiments affected by treatment with DTT and H2O2 (AMV1435)? In this experiment, liver samples were used to test the detrimental effects of treatment with DTT and H2O2 in smFISH experiments. Kidney and brain samples adhere well to the surface without any treatment with DTT and H2O2. This sample was used to investigate the detrimental effects of DTT / H2O2 treatment in FISH experiments. All samples are treated in the same way with respect to rehydration, hybridization, staining and washing steps. In some chambers, combined DTT / H2O2 treatment was performed. Chambers 1-4 served as controls. For details and results, see Table 4 below.

[0067] Table 4: Conditions and results [Table 4]

[0068] As shown in Table 4, the number of spot signals is not significantly affected by treatment with DTT and H2O2. The slight variation in spot signals comes from biological variations within different sections and regions used in the assay.

[0069] 5) Experiment: Is the sensitivity of smFISH experiments affected by treatment with DTT and H2O2 (AMV1435)? In this experiment, liver samples were used to test the detrimental effects of treatment with DTT and H2O2 in smFISH experiments. Liver samples adhere well to surfaces without any treatment with DTT and H2O2. This sample was used to investigate the detrimental effects of DTT / H2O2 treatment in FISH experiments. All samples are treated in the same way with respect to rehydration, hybridization, staining and washing steps. In some chambers, combined DTT / H2O2 treatment was performed. Chambers 1-4 served as controls. For details and results, see Table 5 below.

[0070] Table 5: Conditions and results [Table 5]

[0071] As shown in Table 5, the number of spot signals is not significantly reduced by treatment with DTT and H2O2.

Claims

1. 1. A method for mounting a biological sample on a surface, comprising: i) applying a biological sample to a surface ii) contacting the biological sample on the surface with a reducing agent; and then iii) contacting the biological sample on the surface with an oxidizing agent. A method comprising:

2. The reducing agent is beta-mercaptoethanol, dithiothreitol (DTT), dithioerythritol, sodium borohydride (NaBH ４ ), lithium aluminum hydride (LiAlH ４ ), or another reducing agent of the galvanic series.

3. The oxidizing agent is hydrogen peroxide (H ２ O ２ ), an organic peroxide such as tert-butyl hydroperoxide, sodium perborate, potassium permanganate, hypochlorite, or another oxidizing reagent of the galvanic series.

4. 1. A method for mounting tissue on a slide and for microscopy, comprising: - applying the tissue to the surface of the slide; - contacting the tissue on the slide with a solution of a reducing agent; - drying the slide to remove excess solvent; - contacting the tissue on the slide with an aqueous solution of an oxidizing agent; - microscopic examination of the tissue on the slide and microscopy.

5. The method of claim 4 , wherein the slide comprises a glass, plastic, metal and / or quartz surface.

6. 5. The method of claim 4, wherein the surface is flat or rounded, smooth or rough, hydrophilic or oleophilic.

7. The method of claim 4 , wherein the surface is modified or coated to include additional functionality.

8. 5. The method of claim 4, wherein the tissue is treated with an enzymatic, chemical, or physical treatment or a combination thereof before being applied to the surface.

9. 5. The method of claim 4, wherein the tissue is cut into tissue sections by a microtome or cryotome before being applied to the surface.

10. The method of claim 4 , wherein the tissue comprises lung tissue or skin tissue.

11. The method of claim 4, wherein the tissue comprises biomolecules containing at least disulfide bridges, in particular oxidized thiol groups.

12. The method of claim 4 , wherein the tissue is incubated in a medium or solution before being applied to the surface.

13. 5. The method of claim 4, wherein the tissue is a formalin-fixed, paraffin-embedded (FFPE) tissue.

14. 14. The method of claim 13, wherein the formalin-fixed, paraffin-embedded (FFPE) tissue is deparaffinized after being applied to the surface, in particular by one or more enzymatic treatments, one or more chemical treatments, one or more physical treatments, or a mixture thereof.

15. The reducing agent is beta-mercaptoethanol, dithiothreitol (DTT), dithioerythritol, sodium borohydride (NaBH ４ ), lithium aluminum hydride (LiAlH ４ 15. The method of any one of claims 4 to 14, wherein the oxidizing agent is selected from the group consisting of hydrogen peroxide (H 2 O 2 ), an organic peroxide such as tert-butyl hydroperoxide, sodium perborate, potassium permanganate, hypochlorite, or another oxidizing reagent of the galvanic series.