NeeFISH whole brain tissue transparentizing multiple fluorescence in situ hybridization method and kit

Abstract

The invention discloses a eeFISH (fluorescence in situ hybridization) whole brain tissue transparentizing multiple fluorescence in situ hybridization method and a kit. The hybridization method comprises the steps of whole brain tissue perfusion fixation, decoloration, nucleic acid anchoring, hydrogel crosslinking, electrophoresis-driven transparentizing, probe permeation through osmotic pressure regulation, electrophoresis hybridization and the like. The method specifically comprises the following steps: 1) carrying out covalent anchoring on nucleic acid through a melphalan-succinimide ester compound, and carrying out three-dimensional cross-linking immobilization with an acrylamide-bisacrylamide hydrogel system so as to maintain tissue molecule information and a space structure; (2) molecules such as lipid are removed through electrophoresis, and rapid tissue transparentizing is achieved; and 3) promoting the probe to permeate into the deep layer of the tissue and specifically hybridize under osmotic pressure and electrophoresis conditions. According to the application, high-efficiency electrophoresis driving and osmotic pressure regulation are taken as core characteristics, in-situ detection of whole-brain three-dimensional space molecular information can be directly, efficiently and quickly realized, and the problems of difficulty in compatibility of transparency and hybridization, slow probe permeation, insufficient signal background noise elution and the like are effectively solved; the method is widely applicable to space in-situ detection and analysis of nucleic acid and protein.

Description

Technical Field

[0001] This application belongs to the technical field of tissue transparency multiplex fluorescence in situ hybridization methods, specifically referring to an eeeFISH whole brain tissue transparency multiplex fluorescence in situ hybridization method and a reagent kit. Background Technology

[0002] Exploring the molecular expression information of tissues and organs in three-dimensional space is crucial for accurately understanding the structure, function, and regulatory networks of organisms. Currently, commonly used research methods rely primarily on three-dimensional reconstruction of numerous sequential slices to infer the spatial molecular expression characteristics of tissues and organs. However, this method is not only cumbersome and time-consuming, but also prone to misinterpretation of signals. For example, in studies of axonal regeneration and long-range neuronal projection in the brain, mismatches of discontinuous axons may be mistaken for newly formed axons, and it is difficult to accurately trace the true connection trajectories of distal neuronal projections. Similarly, in physiological and pathological studies of the brain, slice techniques cannot fully reflect the spatial distribution and molecular expression patterns of complex cell types within the brain, potentially leading to biases in the understanding of brain structure and function.

[0003] Therefore, there is an urgent need to develop a method that can directly and accurately detect whole-brain gene expression and spatial information in three-dimensional space. This method is not only crucial for analyzing the structural features and physiological functions of the brain, but also has significant scientific implications for understanding its pathological mechanisms. Summary of the Invention

[0004] The purpose of this application is to provide an eeeFISH (Express, Electrophoretic, and Expansionary Fluorescence in situ Hybridization) method for whole-brain tissue transparency and a reagent kit. This method, characterized by rapid electrophoresis and tissue expansion, effectively integrates tissue transparency with fluorescence in situ hybridization (FISH) through the synergistic effect of efficient electrophoresis-driven and osmotic pressure-regulated approaches. It enables efficient, rapid, and direct in-situ detection and visualization of molecular information on a three-dimensional scale throughout the whole brain. Through electrophoresis-driven and osmotic pressure-regulated processes, the eeeFISH technology system established in this application can rapidly achieve whole-brain tissue transparency, allowing probes and reagents to efficiently and uniformly penetrate deep into the brain tissue for in-situ hybridization. This process significantly improves hybridization efficiency and shortens detection time, overcoming key technical challenges in existing technologies such as incompatibility between transparency conditions and hybridization systems, slow probe penetration speed, and difficulty in completely eluting redundant probes. The establishment of this method provides an important research technique for elucidating brain structure, function, and its molecular regulatory mechanisms.

[0005] In a first aspect, embodiments of this application provide a method for clearing multiplex fluorescence in situ hybridization of whole brain tissue, comprising: Whole brain tissue pretreatment: The isolated whole brain tissue was subjected to perfusion fixation, destaining, nucleic acid anchoring, hydrogel crosslinking and electrophoretic transparentization in sequence; Fluorescence in situ hybridization and imaging: Probe penetration and electrophoretic hybridization, refractive index matching and microscopic imaging were performed sequentially on pretreated whole brain tissue; The perfusion fixation includes: perfusion with a fixation solution to achieve overall fixation of the whole brain tissue; the fixation solution includes a hydrogel embedding solution containing acrylamide and bisacrylamide, and paraformaldehyde; The decolorization includes: using a decolorizing solution to decolorize the perfused and fixed whole brain tissue to remove endogenous pigments; The nucleic acid anchoring includes: reacting melphalan in the nucleic acid anchoring solution with succinimide ester to generate a nucleic acid anchoring complex, wherein the nucleic acid anchoring complex forms a covalent bond with the guanine or adenine bases in nucleic acid molecules in the whole brain tissue, thereby achieving in-situ spatial anchoring of nucleic acid molecules; The hydrogel crosslinking includes: causing the acryloyl terminus carried on the nucleic acid anchoring complex to undergo a crosslinking reaction with the gel crosslinking solution, thereby fixing the anchored nucleic acid molecules in a three-dimensional gel network; the gel crosslinking solution includes acrylamide and bisacrylamide; The electrophoretic-driven transparency process includes: placing the hydrogel-crosslinked whole brain tissue in an electric field, and using a detergent to remove lipid molecules and unanchored nucleic acid molecules from the whole brain tissue during electrophoresis; The probe penetration and electrophoretic hybridization process includes: by adjusting the osmotic pressure and performing electrophoresis, the primary target probe, secondary probe, tertiary probe and signal probe are sequentially driven to penetrate into the whole brain tissue that has been transparentized by electrophoresis; subsequently, each level of probe migrates to the deep layers of the whole brain tissue under the action of electrophoresis and completes specific hybridization within the whole brain tissue; and after the hybridization of each level of probe is completed, it is washed by electrophoresis. The refractive index matching and microscopic imaging process includes: treating whole brain tissue that has undergone probe penetration and electrophoretic hybridization with a refractive index matching solution, followed by microscopic imaging.

[0006] Secondly, embodiments of this application provide a kit comprising: a fixation solution, a decolorization solution, a nucleic acid anchoring solution, a hydrogel crosslinking solution, a crosslinking reaction initiator, a detergent, a primary target probe, a secondary probe, a tertiary probe, a signal probe, and a refractive index matching solution; The fixation solution includes a hydrogel embedding solution containing acrylamide and bisacrylamide, and paraformaldehyde; the nucleic acid anchoring solution includes melphalan and succinimide ester; and the gel crosslinking solution includes acrylamide and bisacrylamide.

[0007] The probe design principle of this application is as follows: To address the technical challenges of incompatible transparency and hybridization conditions, difficulty in probe penetration into the deep layers of the whole brain, slow penetration speed, and difficulty in removing redundant probes in existing technologies, this application provides a method for whole-brain transparency multiplex fluorescence in situ hybridization based on efficient electrophoresis-driven and osmotic pressure-regulated treatment. The technical principle of this method is as follows: 1) Pretreatment of whole-brain tissue: First, a composite system of acrylamide / bisacrylamide and paraformaldehyde is used for perfusion fixation to stabilize tissue morphology and cell structure. Subsequently, decolorization is performed using hydrogen peroxide, ethylenediamine, or amino alcohol solutions to remove endogenous pigments and improve tissue transparency. Next, a complex formed by the reaction of melphalan and succinimide ester is used to achieve covalent anchoring of nucleic acid molecules: the dichloroethylamide group of melphalan forms a covalent addition bond with the nitrogen atom of guanine (G) or adenine (A) in the nucleic acid base; while the acrylamide end of succinimide ester is cross-linked with the hydrogel system, so that the nucleic acid is stably fixed in a three-dimensional gel scaffold network. Finally, electrophoresis is used to remove lipids and unanchored molecules from the tissue, achieving rapid tissue transparency; 2) Electrophoresis-driven fluorescence in situ hybridization: After whole-brain transparency, the tissue is dehydrated by high-osmotic SSC and pretreated with hypotonic hybridization solution to create an active osmotic pressure difference, promoting probe diffusion. Then, a low-voltage electric field is applied to both ends of the electrophoresis system carrying the tissue, causing the nucleic acid probes to migrate directionally into the deep tissue layers under electrophoretic action, achieving rapid and uniform hybridization, thus enabling efficient and rapid multiplex in situ molecular detection of whole-brain transparency.

[0008] The beneficial effects of this application are: Compared with the prior art, this application has the following significant advantages and beneficial effects: (1) This application combines efficient electrophoresis-driven and osmotic pressure regulation strategies to achieve direct and accurate in-situ detection of whole-brain molecular information on a complete three-dimensional spatial scale. This method effectively solves the technical bottleneck of traditional tissue slicing or reconstruction methods, which make it difficult to directly obtain the spatial characteristics of gene expression in three-dimensional structures, and greatly improves spatial resolution and detection structural integrity.

[0009] (2) The hybridization speed of this application is fast. It adopts the synergistic effect of low voltage electrophoresis field driving and high osmotic pressure gradient, so that the molecular probe can penetrate into the deep layer of the whole brain tissue evenly in a short time, realize rapid electrophoretic hybridization, and greatly shorten the experimental cycle. (3) This application has high hybridization efficiency and adopts an electrophoresis-driven probe migration method, which can significantly improve the efficiency of probes entering deep tissue layers compared with traditional passive diffusion hybridization, thereby improving the sufficiency and stability of the overall hybridization reaction.

[0010] (4) This application improves the signal-to-noise ratio and removes unbound redundant probes in tissues quickly by electrophoresis washing, effectively reducing background fluorescence interference and significantly improving signal specificity.

[0011] (5) The present application has high compatibility between transparency and hybridization conditions. Through the optimized hydrogel crosslinking system and osmotic pressure control system, it effectively solves the problem of incompatibility between whole brain transparency and hybridization system conditions in the prior art, so that the sample can still maintain good probe hybridization activity and signal stability under transparency conditions. Attached Figure Description

[0012] Figure 1 This is a schematic diagram illustrating the principle of the eeeFISH whole brain tissue clearing multiplex fluorescence in situ hybridization method described in this application; Figure 2 This is the result of electrophoretic-driven transparency achieved by eeeFISH under complete mouse brain nucleic acid molecular anchoring as shown in Example 1 of this application; Figure 3 As shown in Example 2 of this application, eeeFISH directly detects the contents of intact mouse brain tissue. Gad1 The result of in situ hybridization of gene mRNA; Figure 4 Example 3 of this application demonstrates the direct detection of intact mouse brain tissue using eeeFISH. Sst The result of in situ hybridization of gene mRNA; Figure 5 The results of eeeFISH direct detection of the neuronal marker protein NeuN in intact mouse brain tissue, as shown in Example 4 of this application. Detailed Implementation

[0013] The technical solutions of this application will be further described in detail below with reference to specific embodiments, so as to help those skilled in the art to more clearly and comprehensively understand the principles and implementation methods of this application. However, it should be understood that the embodiments described are only used to illustrate the technical concept and implementation path of this application, and do not constitute a limitation on the scope of protection of this application.

[0014] This application provides a method for clearing multiplex fluorescence in situ hybridization of whole brain tissue, including: Whole brain tissue pretreatment: The isolated whole brain tissue was subjected to perfusion fixation, destaining, nucleic acid anchoring, hydrogel crosslinking and electrophoretic transparentization in sequence; Fluorescence in situ hybridization and imaging: Probe penetration and electrophoretic hybridization, refractive index matching and microscopic imaging were performed sequentially on pretreated whole brain tissue; The perfusion fixation includes: perfusion with a fixation solution to achieve overall fixation of the whole brain tissue; the fixation solution includes a hydrogel embedding solution containing acrylamide and bisacrylamide, as well as paraformaldehyde; Decolorization includes: using a decolorizing solution to decolorize perfused and fixed whole brain tissue to remove endogenous pigments; Nucleic acid anchoring includes: reacting melphalan in the nucleic acid anchoring solution with succinimide ester to form a nucleic acid anchoring complex, and the nucleic acid anchoring complex forming a covalent bond with the guanine or adenine bases in nucleic acid molecules in the whole brain tissue to achieve in-situ spatial anchoring of nucleic acid molecules; Hydrogel crosslinking includes: causing the acrylamide end carried on the nucleic acid anchoring complex to undergo a crosslinking reaction with the gel crosslinking solution, thereby fixing the anchored nucleic acid molecules in a three-dimensional gel network; the gel crosslinking solution includes acrylamide and bisacrylamide; Electrophoretic-driven transparency involves placing the cross-linked whole brain tissue in an electric field, and using a detergent to remove lipid molecules and unanchored nucleic acid molecules from the whole brain tissue during electrophoresis. The probe penetration and electrophoretic hybridization process includes: by adjusting the osmotic pressure and performing electrophoresis, the primary target probe, secondary probe, tertiary probe and signal probe are sequentially driven to penetrate into the whole brain tissue that has been transparentized by electrophoresis; subsequently, each level of probe migrates to the deep layers of the whole brain tissue under the action of electrophoresis and completes specific hybridization within the whole brain tissue; after the hybridization of each level of probe is completed, it is washed by electrophoresis. Refractive index matching and microscopic imaging involves processing whole-brain tissue that has undergone probe penetration and electrophoretic hybridization with a refractive index matching solution, followed by microscopic imaging.

[0015] In this embodiment, the pretreatment and fluorescence in situ hybridization of whole brain tissue are performed sequentially as described above. Whole brain tissue is fixed through perfusion; endogenous pigments are removed and tissue transparency is improved through decolorization; nucleic acid molecules are spatially anchored in situ through nucleic acid anchoring; the acryloyl end of the nucleic acid anchoring complex serves as an anchor point, and hydrogel cross-linking firmly anchors the nucleic acid molecules in a three-dimensional gel scaffold, maintaining their spatial position and providing chemical support for subsequent hybridization and amplification; rapid tissue transparency is achieved through electrophoretic-driven transparency; probe penetration allows for efficient penetration into deeper tissue layers; signal amplification is achieved through electrophoretic hybridization; and higher resolution images can be obtained through refractive index matching and microscopic imaging. The hybridization method of this application can efficiently and rapidly achieve multiplex in situ molecular detection of transparent whole brain tissue. Its design principle is illustrated in the diagram below. Figure 1 As shown.

[0016] In some embodiments, the fixation solution comprises 0.01 to 0.05 g / mL of acrylamide, 0.001 to 0.005 g / mL of bisacrylamide, and 0.04 g / mL of paraformaldehyde (PFA).

[0017] In some embodiments, the fixation time during the infusion fixation process is 5 to 10 minutes.

[0018] In some embodiments, the decolorizing solution includes one or two of hydrogen peroxide and organic amines; the organic amines include one or two of ethylenediamine and amino alcohols. During the decolorization process, whole brain tissue is immersed in the decolorizing solution at 4°C for 8–10 h.

[0019] In some embodiments, the nucleic acid anchoring solution comprises 1–3 mg / mL melphalan and 5–10 mg / mL succinimidyl ester. During the nucleic acid anchoring process, the nucleic acid anchoring complex is prepared by reacting 1–3 mg / mL melphalan with 5–10 mg / mL succinimidyl ester at 4°C with gentle shaking for 5–8 h. The nucleic acid anchoring complex is then incubated with the tissue sample at 37°C for 8–10 h to achieve spatial anchoring of the nucleic acid.

[0020] In some embodiments, the hydrogel crosslinking solution comprises: 0.05–0.1 g / mL sodium acrylate, 0.01–0.05 g / mL acrylamide, 0.001–0.005 g / mL bisacrylamide, 1–2 M sodium chloride, and 1 × PBS buffer.

[0021] In some embodiments, during the hydrogel crosslinking process, the hydrogel crosslinking solution and the nucleic acid anchoring complex undergo a crosslinking reaction under the action of a crosslinking reaction initiator, completing gelation; wherein the crosslinking reaction initiator includes 0.001–0.005 g / mL piperidinol oxide, 0.1–0.2% (V / V) tetramethylethylenediamine, and 0.001–0.002 g / mL ammonium persulfate. The crosslinking reaction temperature is 37 °C, and the reaction time is 8–10 h.

[0022] In some embodiments, the detergent is an anionic detergent or a nonionic detergent. Anionic detergents may include sodium dodecyl sulfate (SDS) (e.g., an anionic detergent containing 0.005–0.02 g / mL SDS); nonionic detergents may include Triton X-100 (e.g., a nonionic detergent containing 0.05–0.2% (V / V) Triton X-100).

[0023] In an optional embodiment, electrophoretic-driven transparency includes: placing the hydrogel-crosslinked whole-brain tissue in an electric field, applying a voltage of 30–60 V to both ends of the electrophoresis system supporting the tissue, and performing electrophoretic removal using an anionic detergent containing 0.005–0.02 g / mL SDS for 20–30 min each time, for a total of 1–3 times. Since lipids (such as phospholipids, sphingomyelin, and cholesterol) in the brain tissue are the main source of light scattering in the electrophoresis tank, electrophoresis allows lipids to form charged complexes with the anionic detergent and migrate along the direction of the electric field, thereby removing lipids, proteins, and unanchored small molecules (e.g., unanchored nucleic acid molecules) from the whole-brain tissue, thus rapidly achieving transparency of the whole-brain tissue.

[0024] In optional embodiments, electrophoretic-driven clearing includes: placing the hydrogel-crosslinked whole-brain tissue in an electric field; applying a voltage of 30–60 V or less to both ends of the electrophoresis system supporting the tissue; and performing electrophoretic removal using a nonionic detergent containing 0.05–0.2% (V / V) Triton X-100 for 20–30 min each time, for a total of 1–3 times. Using a nonionic surfactant system containing 0.05–0.2% (V / V) Triton X-100 and treating at a voltage of 30–60 V or less (e.g., 10–60 V) preferentially removes lipids and unanchored small molecules while maximizing the retention of proteins within the tissue for subsequent detection. In other embodiments, a protease inhibitor may be used to further reduce protein loss and maintain epitope stability.

[0025] In some embodiments, probe penetration includes the following steps: Whole brain tissue that has undergone electrophoretic transparency treatment was placed in 5-6 × SSC solution for a period of time. Discard the 5-6 × SSC solution and add the hybridization probe dissolved in 1-2 × SSC solution to the whole brain tissue, and equilibrate for 10-20 minutes; Add prehybridization solution and apply a voltage of 10–30 V to the electrodes at both ends of the electrophoresis system carrying whole brain tissue for 10–20 minutes to drive the hybridization probe to penetrate deep into the whole brain tissue.

[0026] During probe penetration, electrophoresis can further promote the rapid and uniform penetration of the probe into the deep tissue along the direction of the electric field.

[0027] In some embodiments, during electrophoretic hybridization, for each hybridization stage, a voltage of 10–30 V is applied to both ends of the system containing whole brain tissue and corresponding probe hybridization solution at 40°C for 10–20 minutes, followed by static hybridization for 2–4 hours. After each probe hybridization, the whole brain tissue was transferred to elution buffer at 37°C and washed by electrophoresis with a voltage of 10–20 V. The washing was repeated 3 times, each time for 5–10 minutes.

[0028] The hybridization probes in this embodiment include a primary target probe, a secondary probe, a tertiary probe, and a signal probe. Each level of molecular probe undergoes probe permeation and electrophoretic hybridization sequentially. That is, the primary target probe is first subjected to probe permeation and electrophoretic hybridization, then the secondary probe is subjected to probe permeation and electrophoretic hybridization, then the tertiary probe is subjected to probe permeation and electrophoretic hybridization, and finally the signal probe is subjected to probe permeation and electrophoretic hybridization.

[0029] In some embodiments, the refractive index of the refractive index matching solution is 1.3 to 1.6. In other embodiments, the refractive index matching solution comprises: 0.3 to 0.5 g / mL of sucrose, 0.2 to 0.3 g / mL of urea, 10 to 20% (v / v) of triethanolamine, and 0.1 to 0.5% (v / v) of Triton X-100.

[0030] In some embodiments, DAPI staining may also be used before treating the electrophoretically hybridized and washed whole brain tissue with a refractive index-matching solution.

[0031] In some embodiments, after the first round of refractive index matching and microscopic imaging, DNase I enzyme is added under electrophoresis-driven conditions to digest the hybridized probe, and the reaction is carried out at 37°C for 1–2 h, followed by electrophoresis at 10–30 V. The electrophoresis can be performed three times, each time for 10–20 min, to thoroughly remove any remaining hybridized probe residue, after which a new hybridization reaction can be performed.

[0032] This application is compatible with multi-round, multi-molecule cyclic detection. It utilizes DNase I electrophoretic digestion to achieve reversible removal of probes, enabling multi-round fluorescent probe hybridization and imaging on the same tissue sample. This allows for cyclic detection and quantitative analysis of multiple genes and molecules throughout the whole brain, thereby achieving multi-round signal cyclic detection and improving detection throughput.

[0033] This application provides a kit comprising: a fixation solution, a decolorization solution, a nucleic acid anchoring solution, a hydrogel crosslinking solution, a crosslinking reaction initiator, a detergent, a primary target probe, a secondary probe, a tertiary probe, a signal probe, and a refractive index matching solution; The fixation solution includes a hydrogel embedding solution containing acrylamide and bisacrylamide, as well as paraformaldehyde; the nucleic acid anchoring solution includes melphalan and succinimide ester; and the gel crosslinking solution includes acrylamide and bisacrylamide.

[0034] This application provides a kit that can be used for transparent multiplex fluorescence in situ hybridization of isolated animal organ tissues, specifically for the transparent multiplex fluorescence in situ hybridization of whole brain tissue provided in any of the above embodiments.

[0035] In some embodiments, the fixation solution comprises 0.01–0.05 g / mL acrylamide, 0.001–0.005 g / mL bisacrylamide, and 0.04 g / mL paraformaldehyde. In a specific embodiment, the fixation solution is prepared as follows: containing 1–5 g acrylamide, 0.1–0.5 g bisacrylamide, and 4 g paraformaldehyde powder, diluted to 100 mL with RNase-free water.

[0036] In some embodiments, the decolorizing solution includes one or two of hydrogen peroxide and organic amines; the organic amines include one or two of ethylenediamine and amino alcohols. In a specific embodiment, the decolorizing solution is prepared as follows: containing at least one of 3-10 mL of 30% hydrogen peroxide, 5-10 mL of ethylenediamine, and 5-10 mL of amino alcohol, and diluted to 100 mL with RNase-free water.

[0037] In some embodiments, the nucleic acid anchoring solution comprises 1–3 mg / mL melphalan and 5–10 mg / mL succinimidyl ester. In a specific embodiment, the nucleic acid anchoring solution is prepared as follows: containing 100–300 mg melphalan and 500–1000 mg succinimidyl ester, diluted to 100 mL with RNase-free water, and reacted for 5–8 h to generate the nucleic acid anchoring complex. The reaction temperature can be 4 °C.

[0038] In some embodiments, the hydrogel crosslinking solution comprises: 0.05–0.1 g / mL sodium acrylate, 0.01–0.05 g / mL acrylamide, 0.001–0.005 g / mL bisacrylamide, 1–2 M sodium chloride, and 1 × PBS buffer. In a specific embodiment, the hydrogel crosslinking solution is prepared as follows: containing 5–10 g sodium acrylate, 1–5 g acrylamide, 0.1–0.5 g bisacrylamide, 5–12 g sodium chloride, and 10 mL of 10 × PBS, and brought to a final volume of 100 mL with RNase-free water.

[0039] In some embodiments, the crosslinking reaction initiator is prepared by: containing 0.1–0.5 g piperidinol oxide, 0.1–0.2 mL tetramethylethylenediamine, and 0.1–0.2 g ammonium persulfate, and diluted to 100 mL with RNase-free water.

[0040] In some embodiments, the detergent is an anionic detergent or a nonionic detergent. Anionic detergents may include sodium dodecyl sulfate (SDS) (e.g., an anionic detergent containing 0.005–0.02 g / mL SDS); nonionic detergents may include Triton X-100 (e.g., a nonionic detergent containing 0.05–0.2% Triton X-100 by volume ratio (V / V)).

[0041] In some embodiments, the anionic detergent formulation system is as follows: containing 0.6 g Tris, 0.31 g boric acid, 0.5–2.0 g sodium dodecyl sulfate (SDS), and 0.0186 g disodium ethylenediaminetetraacetate (EDTA·2Na), and diluted to 100 mL with RNase Free Water; In some embodiments, the nonionic detergent formulation system comprises: 0.6 g Tris, 0.31 g boric acid, 0.05–0.2 mL Triton X-100, 1 × EDTA-free protease inhibitor, and RNase-free water to a final volume of 100 mL.

[0042] In some embodiments, the refractive index of the refractive index matching solution is 1.3 to 1.6. In other embodiments, the refractive index matching solution comprises: 0.3 to 0.5 g / mL of sucrose, 0.2 to 0.3 g / mL of urea, 10 to 20% (v / v) of triethanolamine, and 0.1 to 0.5% (v / v) of Triton X-100.

[0043] In some embodiments, the primary target probe is composed of double-stranded DNA, each DNA including a first bottom region, a first middle region and a first top region; the first bottom region has 16 to 25 bases and is used to complement the target sequence; the first middle region has 6 to 8 bases and has 2 to 5 complementary bases; the first top region has 12 to 16 bases.

[0044] In some embodiments, the secondary probe in the kit includes a second signal amplification region and a second intermediate region; the second intermediate region has 25 to 33 bases and is complementary to the first apical region; the second signal amplification region has 20 to 25 bases.

[0045] In some embodiments, the tertiary probe in the kit includes a third signal amplification region and a third intermediate region; the third intermediate region has 20 to 25 bases, and the third intermediate region is complementary to the second signal amplification region, with 16 to 20 bases in the third signal amplification region.

[0046] In some embodiments, both ends of the signal probe, 3' and 5', are labeled with fluorescent groups. These fluorescent groups include, but are not limited to, Alexa Fluor 488, Alexa Fluor 555, and Alexa Fluor 647.

[0047] In optional embodiments, the kit further includes: multiple prehybridization solutions (e.g., prehybridization solution S1, prehybridization solution S2, prehybridization solution S3) and elution buffer.

[0048] In an optional embodiment, the prehybridization solution S1 is prepared as follows: containing 20-30 mL of 20 × SSC, 20-30 mL of deionized formamide, 5-10 mL of 3% LDS, 1-2 mL of vanadate ribonucleoside complex, 5-10 mL of Denhardt's, 10-20 g of dextran sulfate, and RNase-free water to a final volume of 100 mL.

[0049] In an optional embodiment, the prehybridization solution S2 is prepared as follows: containing 20-25 mL of 20 × SSC, 20-25 mL of deionized formamide, 8-10 mL of 3% LDS, 1-2 mL of vanadate ribonucleoside complex, 5-10 mL of Denhardt's, 10-20 g of dextran sulfate, and RNase-free water to a final volume of 100 mL.

[0050] In an optional embodiment, the prehybridization solution S3 is prepared as follows: containing 20-25 mL of 20 × SSC, 5-10 mL of deionized formamide, 8-10 mL of 3% LDS, 1-2 mL of vanadate ribonucleoside complex, 5-10 mL of Denhardt's, 10-20 g of dextran sulfate, and RNase-free water to a final volume of 100 mL.

[0051] In an optional embodiment, the elution buffer is prepared as follows: containing 5-10 mL of 20 × SSC, 5-10 mL of deionized formamide, 8-10 mL of 3% LDS, 1-2 mL of vanadate ribonucleoside complex, and RNase-free water to a final volume of 100 mL.

[0052] It should be noted that the hybridization method or kit in the embodiments of this application is not only applicable to isolated whole brain tissue, but also to the in situ spatial molecular detection of other isolated whole organ tissues (e.g., intact organs such as heart, liver, intestine, kidney, etc.).

[0053] The hybridization methods or kits described in this application can be used for in-situ spatial detection of nucleic acid and protein molecules. Nucleic acid molecules include, but are not limited to, DNA (such as single-stranded DNA, double-stranded DNA, complementary DNA, etc.) and RNA (such as mRNA, lncRNA, miRNA, etc.); protein molecules include, but are not limited to, those that can bind to specific antibodies or aptamer markers.

[0054] The following specific embodiments provide a more detailed description of this application, but should not be construed as limiting the application. Any modifications or substitutions made to the methods, steps, or conditions of this application without departing from the spirit and substance of this application are within the scope of this application.

[0055] Example 1 Example 1 describes a method for electrophoretic-driven transparency of intact mouse brain nucleic acid molecules using eeeFISH. This method includes the following steps: 1.1 Preprocessing of intact mouse brain tissue samples (1) Three-day-old and six-week-old C57BL / 6 mice were selected and perfused with 1× DEPC-PBS buffer to replace the blood in the brain tissue; the perfusion was performed three times, with each injection volume being 20 mL; (2) A fixation solution was formed by mixing a hydrogel embedding solution composed of acrylamide and bisacrylamide and PFA fixation solution in a 1:1 volume ratio. The fixation solution was used to perfuse and fix the mouse brain to stabilize the tissue structure at the molecular level. The concentrations (mass-volume ratios) of acrylamide, bisacrylamide and PFA in the fixation solution were 0.01-0.05 g / mL, 0.001-0.005 g / mL and 0.04 g / mL, respectively. (3) After fixation, the intact mouse brain is placed in a decolorizing solution for treatment; the decolorizing solution can be 0.9-3% (V / V) hydrogen peroxide, 5-10% (V / V) ethylenediamine or 5-10% (V / V) amino alcohol solution, and soaked at 4 °C for 8-10 h to remove endogenous pigments and improve tissue transparency. (4) After decolorization, wash the mouse brain tissue three times with 1× DEPC-PBS buffer for 5-10 min each time to remove residual decolorizing reagent; (5) After washing, equilibrate the tissue with 20 mM MOPS buffer for 20-30 min to provide a buffer environment for the subsequent nucleic acid anchoring reaction; (6) Next, prepare the nucleic acid anchoring complex: mix 1-3 mg / mL melphalan solution with an equal volume of 5-10 mg / mL succinimide ester solution, and react with gentle shaking at 4 °C overnight; incubate the resulting complex with mouse brain tissue at 37 °C for 8-10 h to achieve spatial covalent anchoring of nucleic acid molecules; (7) Subsequently, the tissue was immersed in a room temperature hydrogel system consisting of 0.05–0.1 g / mL sodium acrylate, 0.01–0.05 g / mL acrylamide, 0.001–0.005 g / mL bisacrylamide, 1–2 M sodium chloride and 1 × PBS for 1–2 h. (8) After removal, the mouse brain was soaked again three times with a freshly prepared 4 °C hydrogel system for 1-2 hours each time to ensure that the monomers were fully penetrated; (9) Finally, add 0.001-0.005 g / mL piperidinol oxide, 0.1-0.2% (V / V) tetramethylethylenediamine (TEMED) and 0.001-0.002 g / mL ammonium persulfate (APS), and react at 37 °C for 8-10 h to complete gelation, thereby achieving stable fixation of nucleic acid molecules in a three-dimensional gel network.

[0056] 1.2 Electrophoretic-driven transparentization of intact mouse brain tissue samples (1) The pretreated complete mouse brain was placed in an electrophoresis tank and immersed in an electrophoresis buffer containing 0.006 g / mL Tris, 0.0031 g / mL boric acid, 0.005–0.02 g / mL SDS, and 0.186 mg / mL EDTA·2Na. (2) Subsequently, a voltage of 30-60 V was applied to the electrodes at both ends of the electrophoresis system carrying the whole brain tissue for electrophoretic removal, each time for 20-30 min, for a total of 1-3 times; under the action of the electric field, lipids and unanchored molecules formed charged complexes and migrated along the direction of the electric field, thereby achieving effective removal of lipids, proteins and unanchored small molecules, making the tissue gradually transparent. (3) After the transparency is completed, the sample is placed in a high refractive index matching solution consisting of 0.3~0.5 g / mL sucrose, 0.2~0.3 g / mL urea, 10~20% (V / V) triethanolamine and 0.1~0.5% (V / V) Triton X-100 to perform refractive index matching, so as to further eliminate light scattering and obtain clear microscopic imaging.

[0057] The results are as follows Figure 2 As shown, the eeeFISH method can achieve rapid and efficient tissue transparency in intact mouse brain samples from 3-day-old and 6-week-old mice under electrophoretic drive, providing ideal tissue conditions for subsequent multiplex fluorescence in situ hybridization detection.

[0058] Example 2 Example 2 demonstrates the direct detection of eeeFISH in intact mouse brain tissue. Gad1 An in situ hybridization method for gene mRNA, comprising the following steps: 2.1 Pretreatment of intact mouse brain tissue samples The pretreatment steps for the intact mouse brain tissue samples used in this embodiment are the same as step 1.1 in Example 1.

[0059] 2.2 Fluorescence in situ hybridization and imaging of intact mouse brain tissue samples The electrophoretic transparentization process of intact mouse brain tissue is the same as steps (1) and (2) in step 1.2 of Example 1; the only difference is that in Example 2, electrophoretic in situ hybridization is performed directly after tissue transparentization, so refractive index matching is not performed at this stage. (1) The intact mouse brain tissue that has been transparent by electrophoresis was first placed in a high concentration of 5-6 × SSC solution for dehydration treatment to remove water from the tissue and enhance permeability; then the SSC solution was discarded, and the hybridization probe was added to the 1-2 × SSC system and equilibrated for 10-20 min so that the tissue could actively absorb the probe solution under hypotonic conditions. (2) Next, proceed with Gad1 For hybridization of the primary target probe of the gene, the primary target probe with a final concentration of 50 nM was added to 10 mL of prehybridization solution S1, mixed thoroughly, and preheated at 40 °C. Then, the prehybridization solution S1 was added to the intact mouse brain tissue to be tested, and electrophoretic hybridization was performed at 40 °C and 10–30 V for 10–20 min each time, followed by standing hybridization for 2–4 h. This process was repeated 2–3 times to ensure that the probe fully penetrates the deep tissue and binds to the target sequence. After the primary target probe hybridization was completed, the prehybridization solution S1 was discarded, and the tissue was washed three times with elution buffer Z1 at 37 °C and 10–20 V for 5–10 min each time to remove unbound probes. This embodiment is used for detection Gad1 The primary target probes of the gene consist of 10 pairs. Each pair of probes contains two parts, left and right, and each part includes three regions: a basal region, a middle region, and a apical region. The specific sequences are as follows: Gad1 Sequence information of the basal regions of the 10 pairs of primary target probes of the gene: The left bottom region of the first pair of probes: 5'-GCCACACCAAGTATCATACGTTGTA-3' (SEQ ID No. 1); The right bottom region of the first pair of probes: 5'-AGTTTTCTGGTGCATCCATGGGCTA-3' (SEQ ID No. 2); The second pair of probes has the following left-side bottom region: 5'-AGGCTATTGGTCCTTTGTAAGAAGC-3' (SEQ ID No. 3); The right bottom region of the second pair of probes: 5'-CGCTCACAAGACGACTCTTCTCTTCA-3' (SEQ ID No. 4); The third pair of probes has the following left-side bottom region: 5'-ACAGGTTGGAGAAGTCGGTCTCTGT-3' (SEQ ID No. 5); The right bottom region of the third pair of probes: 5'-CTTAGCTGGAAGCAGATCTTGAGCA-3' (SEQ ID No. 6); The fourth pair of probes, on the left bottom region: 5'-GTCTACCACTTCCAGCAAGAACTGC-3' (SEQ ID No. 7); The right bottom region of the fourth pair of probes: 5'-GTCTTGCGGACATAGTTGAGGAGTA-3' (SEQ ID No. 8); The fifth pair of probes, on the left bottom edge: 5'-TGGGTGGTGGAAATCCAGAACCTTG-3' (SEQ ID No. 9); The right bottom region of the fifth pair of probes: 5'-CCTTCCATGCCTTCCAGCAACTGGT-3' (SEQ ID No. 10); The left bottom region of the sixth pair of probes: 5'-CAACCAGGATCTGCTCCAGAGACTC-3' (SEQ ID No. 11); The right bottom region of the sixth pair of probes: 5'-CCCGTACTTCAGGGTGTCTCTACAG-3' (SEQ ID No. 12); The lower left region of the seventh pair of probes: 5'-ATCCAAACCAGTAGAGAGCTGGTTG-3' (SEQ ID No. 13); The right bottom region of the seventh pair of probes: 5'-AGCCATTCACCAGCTAAACCAATGA-3' (SEQ ID No. 14); The lower left region of the eighth pair of probes: 5'-GGGTGCAATTTCATATGTGAACATA-3' (SEQ ID No. 15); The right bottom region of the eighth pair of probes: 5'-GTAATCTGTTCCATGAGAACAAACA-3' (SEQ ID No. 16); The left bottom region of the ninth pair of probes: 5'-CCATGATGCTGTACATATTGGATAT-3' (SEQ ID No. 17); The right bottom region of the ninth pair of probes: 5'-TTCTGGGAAGTACTTGTAACGAGCA-3' (SEQ ID No. 18); The left bottom region of the tenth pair of probes: 5'-TGTTCTGAGGTGAAGAGGACCAGTT-3' (SEQ ID No. 19); The right bottom region of the tenth pair of probes: 5'-CGGCTTTCTTTATGGAATAGTGACT-3' (SEQ ID No. 20); Gad1 The sequence information of the middle region of all 10 pairs of primary target probes of the gene is identical: Left middle area: 5'-TGGACTTA-3'; Right middle area: 5'-ATTCACCT-3'; Gad1 The apical region sequence information of all 10 pairs of primary target probes of the gene is identical: Left apex region: 5'-CCTATCGATCGTTC-3' (SEQ ID No. 21); Right apex region: 5'-CTTGCTAGCTATCC-3' (SEQ ID No. 22); (3) Next, hybridization of the secondary probe was performed to amplify the signal. The secondary probe with a final concentration of 100 nM was added to 10 mL of prehybridization solution S2, and after thorough mixing, it was preheated at 40 °C. Subsequently, the preheated prehybridization solution S2 was added to the intact mouse brain tissue sample to be tested, and hybridization was performed under electrophoresis conditions of 40 °C and 10–30 V for 10–20 min each time, and the sample was allowed to stand for 2–4 h to promote the specific binding of the secondary probe to the tip region of the primary probe. After the secondary probe hybridization was completed, the prehybridization solution S2 was discarded, and elution buffer Z1 was added. The sample was washed three times by electrophoresis at 10–20 V at 37 °C for 5–10 min each time to completely remove unbound probes. The sequence information of the secondary probe in the intact mouse brain tissue sample is as follows: Sequence information of the repeating regions at both ends of the secondary probe: 5'-CCGATGCGCAGCAATTCACT-3' (SEQ ID No. 23); Sequence information of the intermediate region of the secondary probe: 5'-GGATAGCTAGCAAGTGAACGATCGATAGG-3' (SEQ ID No. 24); (4) Next, hybridization of the tertiary probe was performed for further signal amplification. The tertiary probe with a final concentration of 200 nM was added to 10 mL of prehybridization solution S3, and after thorough mixing, it was preheated at 40 °C. Subsequently, the preheated prehybridization solution S3 was added to the intact mouse brain tissue sample, and electrophoretic hybridization was performed at 40 °C and 10–30 V for 10–20 min each time, and the sample was allowed to stand for 2–4 h to achieve specific binding of the tertiary probe to the repeat sequences at both ends of the secondary probe. After the tertiary probe hybridization was completed, the prehybridization solution S3 was discarded, and elution buffer Z1 was added. The sample was washed three times by electrophoresis at 37 °C and 10–20 V for 5–10 min each time to remove residual unbound probes. The sequence information of the probes used to detect tertiary probes as complementary to secondary probes in intact mouse brain tissue samples is as follows: Sequence information of the repeating regions at both ends of the tertiary probe: 5'-GCGTGAATAGTCCGATCTGG-3' (SEQ ID No. 25); Sequence information of the intermediate region of the tertiary probe: 5'-AGTGAATTGCTGCGCATCGG-3' (SEQ ID No. 26); (5) Next, the signal probe hybridization was continued to achieve the display and detection of fluorescence signal; the signal probe with a final concentration of 300 nM was added to 10 mL of prehybridization solution S3, and after thorough mixing, it was preheated at 40 °C; then, the preheated prehybridization solution S3 was added to the complete mouse brain tissue sample to be tested, and electrophoretic hybridization was performed at 40 °C and 10-30 V for 10-20 min each time, and the hybridization was allowed to stand for 2-4 h to ensure that the signal probe and the tertiary probe recognition sequence were fully bound; after the signal probe hybridization was completed, the prehybridization solution S3 was discarded, and elution buffer Z1 was added. Electrophoretic washing was performed 3 times at 37 °C and 10-20 V for 5-10 min each time to remove unbound or non-specifically bound probe residues; The signal probe used for detecting the signal in intact mouse brain tissue samples is a matching probe to a tertiary probe. Both ends of the signal probe are modified with the Alexa Fluor 488 fluorescent group, and its sequence information is as follows: 5'-CCAGATCGGACTATTCACGC-3' (SEQ ID No. 27); (6) After the hybridization step is completed, the nuclei of whole brain cells are stained with DAPI to reveal the nuclear structure. Then, the samples are placed in a high refractive index matching solution consisting of 0.3-0.5 g / mL sucrose, 0.2-0.3 g / mL urea, 10-20% (V / V) triethanolamine and 0.1-0.5% (V / V) Triton X-100 and soaked at room temperature for 10-30 min to achieve tissue transparency and optical refractive index matching.

[0060] Experimental results are as follows Figure 3 As shown, it is possible to clearly observe in the whole-brain transparent sample. Gad1 Spatial distribution signal of gene mRNA. This embodiment demonstrates that the eeeFISH technology of this application can directly achieve multiplex fluorescence in situ hybridization molecular detection of tissues with transparency at the level of intact tissues and organs.

[0061] In Example 2, the prehybridization solution S1 was prepared as follows: containing 20-30 mL of 20 × SSC, 20-30 mL of deionized formamide, 5-10 mL of 3% LDS, 1-2 mL of vanadate ribonucleoside complex, 5-10 mL of Denhardt's, 10-20 g of dextran sulfate, and RNase-free water to a final volume of 100 mL. The prehybridization solution S2 was prepared as follows: containing 20-25 mL of 20 × SSC, 20-25 mL of deionized formamide, 8-10 mL of 3% LDS, 1-2 mL of vanadate ribonucleoside complex, 5-10 mL of Denhardt's, 10-20 g of dextran sulfate, and RNase-free water to a final volume of 100 mL. The prehybridization solution S3 was prepared as follows: containing 20-25 mL of 20 × SSC, 5-10 mL of deionized formamide, 8-10 mL of 3% LDS, 1-2 mL of vanadate ribonucleoside complex, 5-10 mL of Denhardt's, 10-20 g of dextran sulfate, and RNase-free water to a final volume of 100 mL. The elution buffer Z1 is prepared as follows: containing 5-10 mL of 20 × SSC, 5-10 mL of deionized formamide, 8-10 mL of 3% LDS, 1-2 mL of oxyvanadate ribonucleoside complex, and RNase-free water to a final volume of 100 mL.

[0062] Example 3 Example 3 demonstrates the direct detection of eeeFISH in intact mouse brain tissue. Sst An in situ hybridization method for gene mRNA, comprising the following steps: 3.1 Preprocessing of intact mouse brain tissue samples In this embodiment, the pretreatment steps for the intact mouse brain tissue sample are the same as those in step 2.1 of Example 2; that is, through perfusion, fixation, hydrogel crosslinking and electrophoresis-driven transparency, a transparent intact brain tissue sample suitable for in situ hybridization is obtained.

[0063] 3.2 Fluorescence in situ hybridization and imaging of intact mouse brain tissue samples Direct detection of intact mouse brain tissue Sst The in situ hybridization steps for gene mRNA are basically the same as those in step 2.2 of Example 2; the difference is that: firstly, the probes for the primary target genes are different, requiring the addition of specific targeting probes. Sst Firstly, the primary target probe of the gene; secondly, due to Sst Different fluorescence signals are used for gene detection; therefore, the sequences of the secondary probe, tertiary probe, and signal probe used in this embodiment are different from those in Example 2. Specifically, the probes used in this embodiment for detection... Sst The primary target probe of a gene consists of 5 pairs. The sequence information of the primary target probe, secondary probe, tertiary probe, and signal probe is as follows: Primary target probe sequence information: Sst Sequence information of the basal regions of the five primary target probes of the gene: The left bottom region of the first pair of probes: 5'-CCATTAGCCTCGCAAAATCAATCAC-3' (SEQ ID No. 28); The right bottom region of the first pair of probes: 5'-CCAGATCTCACCAGTGCTTTTACGC-3' (SEQ ID No. 29); The second pair of probes has the following left-side bottom region: 5'-GCTATGGAGCTCTCCACGGTCTCC-3' (SEQ ID No. 30); The right bottom region of the second pair of probes: 5'-GGCTTCGGTAGCGTCTCCTTCAGC-3' (SEQ ID No. 31); The third pair of probes has the following left bottom region: 5'-TTCCTTGCCTCAGGCAGCCAAGCTG-3' (SEQ ID No. 32); The right bottom region of the third pair of probes: 5'-CGCACTGGAGACGGCAGGACAGCAT-3' (SEQ ID No. 33); The fourth pair of probes has the following left bottom region: 5'-AGTACTTGGCCAGTTCCTGTTTCCC-3' (SEQ ID No. 34); The right bottom region of the fourth pair of probes: 5'-GCTCGGACAGCAGCTCTGCCAAG-3' (SEQ ID No. 35); The fifth pair of probes has the following left bottom region: 5'-CTCATCTCGTCCTGCTCAGCTGCCT-3' (SEQ ID No. 36); The right bottom region of the fifth pair of probes: 5'-AGTTGGCAGACCTCTGCAGCTCCAG-3' (SEQ ID No. 37); Sst The sequence information of the middle region of all five pairs of primary target probes of the gene is identical: Left middle area: 5'-TGGACTTA-3'; Right middle area: 5'-ATTCACCT-3'; Sst The sequence information of the apical regions of the five primary target probes of gene 5 is identical: Left apex region: 5'-GTTAGGGACGCTCG-3' (SEQ ID No. 38); Right apex region: 5'-GCTCGCAGGGATTG-3' (SEQ ID No. 39); Secondary target probe sequence information: Sequence information of the repeating regions at both ends of the secondary probe: 5'-CGTCCACGGTGTCAAACAGA-3' (SEQ ID No. 40); Sequence information of the intermediate region of the secondary probe: 5'-CAATCCCTGCGAGCTCGAGCGTCCCTAAC-3' (SEQ ID No. 41); Tertiary target probe sequence information: Sequence information of the repeating regions at both ends of the tertiary probe: 5'-AGGTTAAGCGTTGCGATTAC-3' (SEQ ID No. 42); Sequence information of the intermediate region of the tertiary probe: 5'-TCTGTTTGACACCGTGGACG-3' (SEQ ID No. 43); Signal probe sequence information (both ends of the signal probe are modified with Alexa Fluor 555 fluorescent groups): 5'-GTAATCGCAACGCTTAACCT-3' (SEQ ID No. 44).

[0064] like Figure 4As shown, it can be clearly observed in the transparent, intact mouse brain tissue. Sst The spatial distribution signals of gene mRNA indicate that different fluorescence strategies can effectively detect different RNAs in intact mouse brain tissue, demonstrating the feasibility of eeeFISH for multi-channel detection.

[0065] In addition, the prehybridization solution and elution buffer in this embodiment are the same as those in Example 2.

[0066] Example 4 Example 4 describes a method for directly detecting the neuronal marker protein NeuN in intact mouse brain tissue using eeeFISH. This method includes the following steps: 4.1 Pretreatment of intact mouse brain tissue samples In this embodiment, the pretreatment steps for the complete mouse brain tissue sample are the same as those in step 1.1 of Example 1; that is, through perfusion fixation, hydrogel embedding and nucleic acid anchoring, a brain tissue sample with a complete structure and suitable for electrophoresis is obtained.

[0067] 4.2 Procedure for protein detection in intact mouse brain tissue samples (1) Electrophoresis buffer preparation and protein protection: The pretreated intact mouse brain tissue was placed in the electrophoresis tank and immersed in the electrophoresis buffer without anionic detergent to maximize the protection of the protein structure in the tissue; the composition of the electrophoresis buffer is as follows: containing 0.6 g Tris, 0.31 g boric acid, 0.05-0.2 mL Triton X-100, 1 × EDTA-free protease inhibitor, and adjusted to 100 mL with RNase-free water; (2) Electrophoresis-driven tissue transparency: A voltage of 30-60V is applied to the electrodes at both ends of the electrophoresis system carrying the whole brain tissue for electrophoretic removal, each time for 20-30 min, for a total of 1-3 times; under the action of the electric field, lipids and unanchored small molecules form charged complexes and migrate along the direction of the electric field, thereby effectively removing lipids and non-covalently bound components; at the same time, the amino groups on the lysine residues and the thiol groups on the cysteine ​​residues in the protein can form covalent bonds with the hydrogel matrix, thereby achieving tissue transparency while preserving the protein structure; (3) Tissue sealing: The brain tissue samples that have been made transparent by electrophoresis are placed in a sealing solution for sealing; (4) The blocking solution was prepared by mixing 5% (V / V) donkey serum with 0.3% (V / V) Triton X-100 in 1× PBS solution; electrophoresis was performed at 37 ℃ and 10-30 V for 5-10 min, and then the solution was allowed to stand for 20-30 min to block non-specific binding sites. (5) Primary antibody incubation: After discarding the blocking solution, the cleared intact mouse brain tissue was placed in rabbit-derived NeuN antibody incubation solution; the antibody incubation solution consisted of 1.5% (V / V) donkey serum and 0.1% (V / V) Triton X-100 PBS solution diluted with NeuN primary antibody at a ratio of 1:1000; then, electrophoresis was performed at room temperature at 10-30 V for 5-10 min to promote deep penetration of the antibody, and incubated at 4 ℃ for 12-18 h to achieve full binding of the primary antibody to the target protein; (6) Primary antibody washing: After incubation, place the sample in PBST solution containing 0.05% (V / V) Tween-20 and electrophoretically wash 3 to 4 times at room temperature and 10 to 30 V voltage for 5 to 10 min each time to remove unbound primary antibody molecules; (7) Secondary antibody incubation: Place brain tissue samples in donkey anti-rabbit secondary antibody incubation solution diluted 1:1000 (diluted with PBS), apply 10-30 V voltage for electrophoresis at room temperature for 5-10 min, and incubate at 37 ℃ for 1-2 h to promote full binding of fluorescent secondary antibody; (8) Secondary antibody washing: After incubation, place the sample in PBST solution containing 0.05% (V / V) Tween-20 and electrophoretically wash 3 to 4 times at room temperature and 10 to 30 V voltage for 5 to 10 min each time to remove excess unbound fluorescent secondary antibody; (9) Cell nuclear staining and refractive index matching: The sample is stained with DAPI to reveal the cell nuclear structure; then the sample is immersed in a high refractive index matching solution (composed of 0.3-0.5 g / mL sucrose, 0.2-0.3 g / mL urea, 10-20% (V / V) triethanolamine and 0.1-0.5% (V / V) Triton X-100) at room temperature for 10-30 min to achieve optical refractive index matching of the tissue. After transparency and refractive index adjustment, three-dimensional light film or confocal imaging can be performed directly.

[0068] like Figure 5 As shown, NeuN protein signals are clearly visible in transparent, intact mouse brain tissue. The results indicate that the eeeFISH technique can not only detect nucleic acid molecular signals at the intact tissue scale, but also achieve high-fidelity spatial detection of whole-brain proteins while preserving the tissue protein structure. This verifies the broad applicability of this method in nucleic acid-protein co-detection and whole-brain imaging.

[0069] In summary, the embodiments disclosed in this application are only used to illustrate the technical solutions and implementation methods of this application, and are not intended to limit the scope of protection of this application. For those skilled in the art, various adjustments, equivalent substitutions, functional transformations, or structural improvements to the technical features made without departing from the core ideas of this application should be considered to fall within the scope of protection claimed in this application.

Claims

1. A method for clearing whole brain tissue using multiplex fluorescence in situ hybridization, characterized in that, include: Whole brain tissue pretreatment: The isolated whole brain tissue was subjected to perfusion fixation, decolorization, nucleic acid anchoring, hydrogel crosslinking and electrophoretic transparentization in sequence; Fluorescence in situ hybridization and imaging: Probe penetration and electrophoretic hybridization, refractive index matching and microscopic imaging were performed sequentially on pretreated whole brain tissue; The perfusion fixation includes: perfusion with a fixation solution to achieve overall fixation of the whole brain tissue; the fixation solution includes a hydrogel embedding solution containing acrylamide and bisacrylamide, and paraformaldehyde; The decolorization includes: using a decolorizing solution to decolorize the perfused and fixed whole brain tissue to remove endogenous pigments; The nucleic acid anchoring includes: reacting melphalan in the nucleic acid anchoring solution with succinimide ester to generate a nucleic acid anchoring complex, wherein the nucleic acid anchoring complex forms a covalent bond with the guanine or adenine bases in nucleic acid molecules in the whole brain tissue, thereby achieving in-situ spatial anchoring of nucleic acid molecules; The hydrogel crosslinking includes: causing the acryloyl terminus carried on the nucleic acid anchoring complex to undergo a crosslinking reaction with the gel crosslinking solution, thereby fixing the anchored nucleic acid molecules in a three-dimensional gel network; the gel crosslinking solution includes acrylamide and bisacrylamide; The electrophoretic-driven transparency process includes: placing the hydrogel-crosslinked whole brain tissue in an electric field, and using a detergent to remove lipid molecules and unanchored nucleic acid molecules from the whole brain tissue during electrophoresis; The probe penetration and electrophoretic hybridization process includes: by adjusting the osmotic pressure and performing electrophoresis, the primary target probe, secondary probe, tertiary probe and signal probe are sequentially driven to penetrate into the whole brain tissue that has been transparentized by electrophoresis; subsequently, each level of probe migrates to the deep layers of the whole brain tissue under the action of electrophoresis and completes specific hybridization within the whole brain tissue; and after the hybridization of each level of probe is completed, it is washed by electrophoresis. The refractive index matching and microscopic imaging process includes: treating whole brain tissue that has undergone probe penetration and electrophoretic hybridization with a refractive index matching solution, followed by microscopic imaging.

2. The hybridization method according to claim 1, characterized in that, The fixative solution comprises 0.01~0.05 g / mL acrylamide, 0.001~0.005 g / mL bisacrylamide, and 0.04 g / mL paraformaldehyde; And / or, the decolorizing solution comprises one or two of hydrogen peroxide and organic amines; the organic amine comprises one or two of ethylenediamine and amino alcohols; And / or, the nucleic acid anchoring solution comprises 1–3 mg / mL melphalan and 5–10 mg / mL succinimide ester; And / or, the hydrogel crosslinking solution comprises: 0.05–0.1 g / mL sodium acrylate, 0.01–0.05 g / mL acrylamide, 0.001–0.005 g / mL bisacrylamide, 1–2 M sodium chloride, and 1 × PBS buffer; And / or, the detergent is an anionic detergent or a nonionic detergent.

3. The hybridization method according to claim 1 or 2, characterized in that, During the hydrogel crosslinking process, the hydrogel crosslinking solution undergoes a crosslinking reaction with the nucleic acid anchoring complex under the action of the crosslinking reaction initiator; The crosslinking reaction initiator includes 0.001–0.005 g / mL piperidinol oxide, 0.1–0.2% (V / V) tetramethylethylenediamine, and 0.001–0.002 g / mL ammonium persulfate.

4. The hybridization method according to claim 1 or 2, characterized in that, The electrophoretic-driven transparency process includes: placing the hydrogel-crosslinked whole brain tissue in an electric field, applying a voltage of 30–60 V to both ends of the electrophoresis system supporting the tissue, and performing electrophoretic removal using an anionic detergent containing 0.005–0.02 g / mL SDS for 20–30 min each time, for a total of 1–3 times, to remove lipids, proteins, and unanchored nucleic acid molecules from the whole brain tissue, thereby rapidly achieving transparency of the whole brain tissue.

5. The hybridization method according to claim 1 or 2, characterized in that, The electrophoretic-driven transparency process includes: placing the hydrogel-crosslinked whole brain tissue in an electric field, applying a voltage of 10–60 V to both ends of the electrophoresis system supporting the tissue, and performing electrophoretic removal using a non-ionic detergent containing 0.05–0.2% (V / V) Triton X-100 for 20–30 minutes each time, for a total of 1–3 times, to remove lipids and unanchored nucleic acid molecules in the whole brain tissue while retaining proteins in the whole brain tissue.

6. The hybridization method according to claim 1 or 2, characterized in that, The probe penetration includes: Whole brain tissue that has undergone electrophoretic transparency treatment was placed in 5-6 × SSC solution for a period of time. Discard the 5-6 × SSC solution, add the hybridization probe dissolved in 1-2 × SSC solution to the whole brain tissue, and equilibrate for 10-20 minutes; Add prehybridization solution and apply a voltage of 10–30 V to the electrodes at both ends of the electrophoresis system carrying whole brain tissue for 10–20 minutes to drive the hybridization probe to penetrate deep into the whole brain tissue.

7. The hybridization method according to claim 1 or 2, characterized in that, During the electrophoretic hybridization process, for each hybridization stage, a voltage of 10-30 V is applied to the electrodes at both ends of the electrophoresis system containing whole brain tissue and corresponding probe hybridization solution at 40°C for 10-20 minutes, followed by static hybridization for 2-4 hours. After each probe hybridization, the whole brain tissue was transferred to an elution buffer at 37°C and washed by electrophoresis at a voltage of 10–20 V. The washing was repeated three times, each time for 5–10 minutes.

8. The hybridization method according to claim 1 or 2, characterized in that, The refractive index of the refractive index matching solution is 1.3 to 1.6; And / or, the refractive index matching solution comprises: 0.3 g / mL to 0.5 g / mL of sucrose, 0.2 g / mL to 0.3 g / mL of urea, 10 to 20% (V / V) of triethanolamine and 0.1 to 0.5% (V / V) of Triton X-100.

9. The hybridization method according to claim 1 or 2, characterized in that, After the first round of refractive index matching and microscopic imaging, DNase I enzyme was added under electrophoresis to digest the hybridized probes, and the reaction was carried out at 37°C for 1-2 h. Then, electrophoresis was carried out at 10-30 V to remove the hybridization probes from the first round, so as to facilitate the next round of hybridization reaction.

10. A reagent kit, characterized in that, include: Fixation solution, decolorization solution, nucleic acid anchoring solution, hydrogel crosslinking solution, crosslinking reaction initiator, detergent, primary target probe, secondary probe, tertiary probe, signal probe, refractive index matching solution; The fixation solution includes a hydrogel embedding solution containing acrylamide and bisacrylamide, and paraformaldehyde; the nucleic acid anchoring solution includes melphalan and succinimide ester; and the gel crosslinking solution includes acrylamide and bisacrylamide.

11. The reagent kit according to claim 10, characterized in that, The fixative solution comprises 0.01~0.05 g / mL acrylamide, 0.001~0.005 g / mL bisacrylamide, and 0.04 g / mL paraformaldehyde; And / or, the decolorizing solution comprises one or two of hydrogen peroxide and organic amines; the organic amine comprises one or two of ethylenediamine and amino alcohols; And / or, the nucleic acid anchoring solution comprises 1–3 mg / mL melphalan and 5–10 mg / mL succinimide ester; And / or, the hydrogel crosslinking solution comprises: 0.05–0.1 g / mL sodium acrylate, 0.01–0.05 g / mL acrylamide, 0.001–0.005 g / mL bisacrylamide, 1–2 M sodium chloride, and 1 × PBS buffer; And / or, the detergent is an anionic detergent or a nonionic detergent; And / or, the refractive index of the refractive index matching solution is 1.3 to 1.

6.

12. The reagent kit according to claim 11, characterized in that, The anionic detergent includes sodium dodecyl sulfate; the nonionic detergent includes Triton X-100.

13. The reagent kit according to claim 10, characterized in that, The fixative solution was prepared as follows: containing 1-5 g acrylamide, 0.1-0.5 g bisacrylamide, and 4 g paraformaldehyde powder, and diluted to 100 mL with RNase-free water. The decolorizing solution is prepared by adding at least one of the following: 3-10 mL of 30% hydrogen peroxide, 5-10 mL of ethylenediamine, and 5-10 mL of amino alcohol, and then adjusting the volume to 100 mL with RNase-free water. The preparation system of the nucleic acid anchoring solution is as follows: containing 100-300 mg melphalan and 500-1000 mg succinimide ester, diluted to 100 mL with RNase-free water, and reacted for 5-8 h to generate a nucleic acid anchoring complex; The hydrogel crosslinking solution was prepared as follows: containing 5–10 g sodium acrylate, 1–5 g acrylamide, 0.1–0.5 g bisacrylamide, 5–12 g sodium chloride, and 10 mL 10 × PBS, and then diluted to 100 mL with RNase-free water. The crosslinking reaction initiator is prepared by: containing 0.1–0.5 g piperidinol oxide, 0.1–0.2 mL tetramethylethylenediamine, and 0.1–0.2 g ammonium persulfate, and diluted to 100 mL with RNase-free water; The detergent is an anionic detergent or a nonionic detergent; The formulation system of the anionic detergent is as follows: containing 0.6 g Tris, 0.31 g boric acid, 0.5–2.0 g sodium dodecyl sulfate (SDS), and 0.0186 g disodium ethylenediaminetetraacetate (EDTA·2Na), and diluted to 100 mL with RNase-free water. The nonionic detergent formulation system consists of 0.6 g Tris, 0.31 g boric acid, 0.05–0.2 mL Triton X-100, 1 × EDTA-free protease inhibitor, and RNase-free water to a final volume of 100 mL.

14. The kit according to claim 10, characterized in that, The primary target probe is composed of double-stranded DNA, each DNA strand including a first bottom region, a first middle region, and a first top region; the first bottom region has 16 to 25 bases and is used to complement the target sequence; the first middle region has 6 to 8 bases and carries 2 to 5 complementary bases; the first top region has 12 to 16 bases. The secondary probe in the kit includes a second signal amplification region and a second intermediate region; the second intermediate region has 25-33 bases and is complementary to the first apical region; the second signal amplification region has 20-25 bases. The kit contains a tertiary probe comprising a third signal amplification region and a third intermediate region; the third intermediate region has 20-25 bases and is complementary to the second signal amplification region, and the third signal amplification region has 16-20 bases. The signal probe has fluorescent groups labeled at both ends of its 3' and 5' ends.

Images