A single-enzyme double-substrate immunohistochemical double staining method without host species restriction of primary antibody

Abstract

The application discloses a single-enzyme double-substrate immunohistochemical double-staining method without host species restriction of antibodies, and belongs to the technical field of immunohistochemical staining.The immunohistochemical double-staining method in the application first carries out antigen repair on pretreated tissue sections, and then carries out first staining; the first staining uses 10% homologous serum of secondary antibodies as a diluent, and uses AEC color developing solution for color development; then, the Tris-EDTA sodium citrate mixed solution is heated to strip the primary and secondary antibodies combined on the section, and second staining is carried out; the second staining uses 10% homologous serum of secondary antibodies containing 0.1% Triton-100 as a diluent, and uses DAB color developing solution containing 0.1% nickel ammonium sulfate for color development; finally, the tissue sections are air-dried, and are mounted with glycerol gelatin.The double-staining method in the application has the advantages of wide applicability, low cross-reactivity, small antigen loss, high positive signal color contrast (red and black), time saving, easy operation and low cost.

Description

Technical Field

[0001] This invention relates to the field of immunohistochemical staining technology, and in particular to a single-enzyme dual-substrate immunohistochemical dual staining method without primary antibody host species limitations. Background Technology

[0002] Immunohistochemical staining is a technique that uses the principle of specific binding between antigens and antibodies, combined with a display agent, to locate and semi-quantitatively analyze antigens within tissue cells. Commonly used display agents include fluorescein, enzymes, metal ions, and isotopes. When it is necessary to observe two or more antigens simultaneously on a single slide, multiplex immunostaining techniques are required.

[0003] Multiplex immunostaining most commonly employs immunofluorescence (IF) and tyramide signal amplification (TSA) techniques. Since both use fluorescein as the display agent, fluorescence imaging is necessary. However, in some cases, fluorescence imaging cannot avoid interference from tissue autofluorescence. Common tissue autofluorescence substances include, but are not limited to: hemoglobin, collagen, melanin, NADH, lipofuscin, riboflavin, and elastin. Specimens that have not been perfused (clinical specimens) or have vascular disturbances (tumors, cerebral hemorrhage) often retain a large number of red blood cells; NADH is an essential enzyme for many metabolic processes and is abundant in metabolically active cells and tissues (e.g., liver); lipofuscin is an age-related pigment deposit that accumulates in many tissue cells, including skeletal muscle, nerve tissue, heart, liver, and skin; and tissues such as skin are rich in collagen and elastin. Furthermore, when specimens are fixed in aldehyde solutions, the aldehyde reacts with amines in a Schiff base reaction, generating autofluorescence. Therefore, when the specimens being tested are rich in autofluorescent substances, including but not limited to liver tissue, skin tissue, neural tissue of mice and rats older than 3 months, clinical specimens, cerebral hemorrhage model specimens, experimental animal tumors, and aldehyde-fixed tissues, the use of immunofluorescence or TSA techniques for multiple labeling inevitably encounters the problem of autofluorescence. Although using fluorescence quenchers such as Sudan Black can suppress the autofluorescence effect to some extent, these quenchers also suppress the signal of the fluorescein being tested, leading to a decrease in sensitivity; laser bleaching technology has a good quenching effect on autofluorescence, but it requires a specialized laser bleaching instrument, which is costly and difficult to popularize.

[0004] In contrast, immunohistochemical enzyme staining using bright-field imaging is not affected by autofluorescence. Immunohistochemical enzyme staining involves labeling antibodies with enzymes, which then react with chromogenic substrates to generate clearly colored precipitates, revealing the location and quantity of antigens. Commonly used enzymes include horseradish peroxidase (HRP) and alkaline phosphatase (AP). Commonly used chromogenic substrates for HRP include: diaminobenzidine (DAB), which produces a brownish-yellow positive reaction; 3-amino-9-ethylcarbazole (AEC), which produces a red positive reaction; and DAB with nickel ion enhancement, producing a black positive reaction. Other substrates suitable for AP include: 5-bromo-4-chloro-3-indole-phosphate / nitrotetrazole blue chloride (BCIP / NBT), which produces a blue positive reaction; and fast red, which produces a red positive reaction. Commonly used immunohistochemical enzyme staining methods include direct and indirect methods. The direct method directly labels the enzyme onto the primary antibody, but commercially available enzyme-labeled primary antibodies have low selectivity. Indirect methods include two-step and three-step methods. The two-step method labels the enzyme onto the secondary antibody, while the three-step method labels the secondary antibody onto biotin, and then labels the enzyme onto avidin and / or streptomycin, which have a high affinity for biotin. The three-step method involves more steps, and the binding specificity of biotin to avidin is lower than that of antigen-antibody reactions.

[0005] Currently, various multiplex staining methods for immunohistochemistry exist; however, these methods still have some drawbacks. Liang Yingjie et al. reported a triple immunoenzymatic histochemical staining method using microwave treatment to elute antibodies, but this method is cumbersome, requires numerous reagents, and is time-consuming. Wang Yongjun et al. reported an HRP dual-substrate (DAB and AEC) immunohistochemical double staining method, using 0.05% HCl to elute bound antibodies, followed by a sodium citrate antigen retrieval before the second round of primary antibody incubation. This method uses acid washing to remove bound antibodies and requires two antigen retrievals, making it cumbersome, and the chosen brown and red colors have low contrast. Henry J Waldvogel et al. reported an IHC double staining method using a combination of DAB nickel ion enhanced staining (black) and DAB staining (brownish-yellow). This method uses primary antibodies from different species, does not involve dedicated antibody elution, and uses a three-step approach. However, the chosen brown-yellow and black colors have low contrast, the steps are cumbersome, and different species of primary antibodies are required. The novel immunoenzyme histochemical multiple staining technique developed by She Guizhou et al., which utilizes computer image processing technology, requires the removal of AECs (washing with 80% alcohol for more than 1 hour) and the elution of bound antibodies in each round of staining (microwave heating with sodium citrate retrieval solution for 20 minutes). Furthermore, the images after each round of staining require manual comparison and overlay using Photoshop software. This method is cumbersome and time-consuming, and it is difficult to avoid relative misalignment when overlapping images. Because only AECs are used for staining throughout the process, each round of staining results in a red color, with only variations in red intensity after different antibody reactions. The synthesized images then require color replacement based on the red intensity, making it difficult to guarantee the accuracy of the replacement. The two HRP substrate sequential staining methods for IHC double staining developed by Kenneth HeeschePetersen et al. can use primary antibodies of the same species throughout the process, but also use acid elution to remove antibodies, making it impossible to completely avoid cross-reactions. Recently, Han Xue et al. reported a method for double staining of antibodies derived from the same species using immunohistochemistry. The first staining uses a two-step HRP / DAB method for color development, with dilute sulfuric acid used to elute the antibody in between. The second staining uses a three-step AP / Firm Red method for color development. However, this method cannot avoid the drawbacks of acid causing antigen loss and cross-reactivity.

[0006] Overall, despite the existence of various multiplex immunostaining methods, some challenges remain. Therefore, exploring a more optimized multiplex immunostaining technique is an important topic in the current field of immunostaining. Summary of the Invention

[0007] To address the aforementioned problems, this invention aims to provide a single-enzyme dual-substrate immunohistochemical double staining method without host species limitations of the primary antibody. This method has the advantages of wide applicability, low cross-reactivity, minimal antigen loss, high contrast of positive signal colors, time-saving and easy to implement, and low cost.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] A single-enzyme dual-substrate immunohistochemical double staining method without host species restrictions of the primary antibody includes the following steps:

[0010] S1: Tissue section pretreatment;

[0011] S2: Use Tris-EDTA antigen retrieval solution to perform antigen retrieval on pretreated tissue sections;

[0012] S3: Perform the first staining using AEC chromogenic solution;

[0013] S4: Debonded primary and secondary antibodies were removed from sections immersed in a Tris-EDTA sodium citrate mixture by heating;

[0014] S5: Perform a second staining using DAB colorimetric solution containing 0.1% nickel ammonium sulfate;

[0015] S6: Seal the film and acquire the image.

[0016] Furthermore, the Tris-EDTA antigen retrieval solution described in step S2 contains 1 mM EDTANa2·2H2O and 10 mM Tris.

[0017] Furthermore, the antigen retrieval method described in step S2 includes the following steps: placing the Tris-EDTA antigen retrieval solution in a microwave oven and preheating it to boiling on high heat, then immersing the pretreated tissue slices in the Tris-EDTA antigen retrieval solution, continuing to heat it to boiling on high heat, immediately turning to medium heat and maintaining it for 10 minutes, and then allowing it to cool naturally to room temperature.

[0018] Furthermore, the method for the first staining in step S3 includes the following steps:

[0019] S301: Incubate with the first primary antibody, then wash with PBS; the first primary antibody dilution is 10% secondary antibody homologous serum.

[0020] S302: Incubate with the first HRP-labeled secondary antibody, then wash with PBS; the first secondary antibody dilution is 10% homologous serum.

[0021] S303: First color development: Add AEC working solution, develop color for 10-30 minutes, positive signal is red, rinse with running water.

[0022] Furthermore, the specific operation of step S4 includes the following steps: immersing the tissue sections after AEC staining in a Tris-EDTA sodium citrate mixture, heating it to boiling on high in a microwave oven, immediately turning to medium heat and maintaining for 10 minutes, then naturally cooling to room temperature, rinsing with running water and then rinsing with distilled water.

[0023] Furthermore, the preparation method of the Tris-EDTA sodium citrate mixture is as follows: add 2mM citric acid to 10mM trisodium citrate dihydrate to prepare sodium citrate working solution, and then mix Tris-EDTA antigen retrieval solution and sodium citrate working solution at a volume ratio of 2:1.

[0024] Furthermore, the second staining operation described in step S5 includes the following steps:

[0025] S501: Incubate with the second primary antibody, then wash with PBS; the second primary antibody dilution solution is 10% homologous serum containing 0.1% Triton-100 secondary antibody.

[0026] S502: Incubate with the second HRP-labeled secondary antibody, then wash with PBS; the second secondary antibody dilution solution is 10% homologous serum containing 0.1% Triton-100.

[0027] S503: Secondary color development; add DAB solution containing 0.1% nickel ammonium sulfate, develop under a microscope for 1-10 minutes, positive signal is black, rinse with running water.

[0028] Furthermore, in step S6, the slide is sealed with glycerin gelatin.

[0029] The beneficial effects of this invention are:

[0030] 1. The immunohistochemical double staining method in this invention has no species restriction on the source of the primary antibody for the target, which greatly improves the applicability of immunohistochemical double staining; if all the primary antibodies come from the same species, then only one corresponding HRP-labeled secondary antibody is needed, which reduces the types of experimental reagents and lowers the detection cost.

[0031] 2. In this invention, during staining, AEC staining (first round) should be performed first, followed by DAB staining with 0.1% nickel ammonium sulfate (second round). The two rounds of staining use two high-contrast colors, red and black, respectively, which optimizes the color source compatibility and enables bright-field visualization of two co-localized antigens.

[0032] 3. The Tris-EDTA sodium citrate mixture heating method used in this invention can efficiently elute the bound antibodies and HRP, avoiding cross-reaction.

[0033] 4. The immunohistochemical double staining method in this invention is particularly suitable for multiple immunostaining of specimens rich in autofluorescent substances, such as liver tissue, skin tissue, nerve tissue of mice and rats older than 3 months, clinical solid specimens, cerebral hemorrhage model specimens, experimental animal tumors, and tissues fixed with paraformaldehyde or formalin.

[0034] 5. The immunohistochemical double staining method in this invention does not require the introduction of the AP colorimetric system. The entire process uses HRP-labeled secondary antibodies. HRP molecules are much smaller than AP molecules and have a lower steric hindrance effect. Therefore, when the two antigens being tested co-localize, the sensitivity of the second HRP colorimetric detection is higher than that of the AP colorimetric system.

[0035] 6. The immunohistochemical double staining method in this invention uses a two-step process for both staining processes, instead of the three-step process that requires the introduction of biotin, thus saving time and ensuring the specificity of the antigen-antibody reaction.

[0036] 7. The immunohistochemical double staining method in this invention uses bright-field imaging, which eliminates the need for expensive fluorescence microscopes, has a low barrier to entry for experimental platforms, and is easy to promote. Attached Figure Description

[0037] Figure 1 This is a bright-field microscopy image obtained after immunohistochemical double staining of glial fibrillary acidic protein (GFAP) / tyrosine hydroxylase (TH) and ionized calcium binding adapter 1 (Iba-1) / TH in mouse brain tissue, treated with 1-methyl-4-phenyl-1,2,3,6-tetrahydropyridine (MPTP).

[0038] Figure 2 This is a bright-field microscopy image of α-synuclein [p-α-synuclein(S129)] phosphorylated with serine at amino acid position 129 of the locus coeruleus in the brain tissue of the SNCA-A53T mutant transgenic mouse brain tissue in Application Example 2 of this invention after double staining with TH immunohistochemistry.

[0039] Figure 3This is a bright-field microscopy image of the corpus callosum of a 10-week-old wild-type C57BL / 6J mouse brain tissue after immunohistochemical double staining for aromatic L-amino acid decarboxylase (AADC) and oligodendrocyte lineage transcription factor 2 (Olig2). This is an example of application of the present invention in Example 3. Detailed Implementation

[0040] To enable those skilled in the art to better understand the technical solutions of the present invention, the technical solutions of the present invention will be further described below in conjunction with the accompanying drawings and embodiments.

[0041] Example 1:

[0042] Example 1 provides a single-enzyme dual-substrate immunohistochemical double staining method for paraffin tissue sections without primary antibody host species restriction, specifically including the following steps:

[0043] S1: Pretreatment of paraffin tissue sections;

[0044] Specifically, S101: Baking slices; bake slices at 65℃ for 1 hour;

[0045] S102: Dewaxing; xylene dewaxing 3 times, 10 minutes each time;

[0046] S103: Hydration; Soak the dewaxed sections in 100% alcohol, 95% alcohol, 85% alcohol and 75% alcohol for 5 minutes in sequence; then rinse with running water for 5 minutes and rinse with distilled water for 3 minutes.

[0047] Further, step S2: Use Tris-EDTA antigen retrieval solution to perform antigen retrieval on the pretreated tissue sections;

[0048] Specifically, first boil the Tris-EDTA antigen retrieval solution over high heat, then immerse the pretreated paraffin tissue section from step S1 in the Tris-EDTA antigen retrieval solution and continue microwave heating. After boiling, reduce the heat to medium and maintain heating for 10 minutes, then turn off the heat. Allow it to cool naturally, rinse with running water for 5 minutes, and then rinse with distilled water for 3 minutes.

[0049] This procedure can also be performed using a pressure cooker to heat-retrieve antigens from pretreated paraffin tissue sections: Immerse the pretreated paraffin tissue sections from step S1 in the Tris-EDTA antigen retrieval solution in the pressure cooker, place the pressure cooker on an electromagnetic induction heater, wait for the steam to come out, then time for 3 minutes, and then turn off the heat; allow it to cool naturally, rinse with running water for 5 minutes, and then rinse with distilled water for 3 minutes.

[0050] The preparation method of Tris-EDTA antigen retrieval solution is as follows: Mix 1mM EDTANa2·2H2O with 10mM Tris evenly to obtain Tris-EDTA (tris(hydroxymethyl)aminomethane, Tris)-ethylenediaminetetraacetic acid (EDTA)) antigen retrieval solution.

[0051] Further, step S3: perform the first staining using AEC staining solution;

[0052] Before performing the first staining, it is necessary to inactivate endogenous peroxidase and block non-specific binding sites.

[0053] Specifically, use a drawing pen to circle the tissue section, add 3% hydrogen peroxide, and incubate at room temperature for 20 minutes; add 10% secondary antibody homologous serum for blocking, and incubate at room temperature for 20 minutes.

[0054] The specific procedures for the first staining include:

[0055] S301: Incubate with the first primary antibody; discard the serum, do not wash, and add the first primary antibody (the first primary antibody dilution solution is 10% secondary antibody homologous serum, incubate at 4℃ for 12-16 hours or at 37℃ for 1 hour); rinse 3 times with PBS, 5 minutes each time;

[0056] S302: Incubate with the first HRP-labeled secondary antibody; add the first secondary antibody (the first secondary antibody dilution solution is 10% homologous serum, incubate at room temperature for 30 min); wash 3 times with PBS, 5 min each time;

[0057] S303: First color development: Add AEC working solution (3-amino-9-ethylcarbazole working solution, freshly prepared and used), develop color for 10-30 minutes, positive signal is red; stop with tap water, rinse with running water for 5 minutes.

[0058] Further, step S4: peeling the bound primary and secondary antibodies from the slices immersed in the Tris-EDTA sodium citrate mixture by heating;

[0059] Specifically, S401: Immerse the tissue sections after AEC staining in a mixture of Tris-EDTA and sodium citrate, heat in a microwave oven to boiling, then reduce to medium heat and maintain for 10 minutes to elute the bound primary and secondary antibodies; allow to cool naturally, rinse with running water for 5 minutes, and rinse with distilled water for 3 minutes.

[0060] The preparation method of the Tris-EDTA sodium citrate mixture is as follows: add 2mM citric acid to 10mM trisodium citrate dihydrate to prepare sodium citrate working solution, and then mix Tris-EDTA antigen retrieval solution with sodium citrate working solution at a volume ratio of 2:1. All working solutions use pure water as solvent.

[0061] S402: Second blocking; add 10% secondary antibody homologous serum for blocking, and incubate at room temperature for 20 min.

[0062] Further, step S5: perform a second staining using DAB colorimetric solution containing 0.1% nickel ammonium sulfate;

[0063] Specifically, S501: Incubate with the second primary antibody; discard the serum, do not wash, add the second primary antibody (the second primary antibody dilution solution is 10% secondary antibody homologous serum containing 0.1% Triton-100, incubate at room temperature for 1.5h), wash 3 times with PBS, 5min each time;

[0064] S502: Incubate the second HRP-labeled secondary antibody; add the second secondary antibody (the second secondary antibody dilution solution is 10% homologous serum containing 0.1% Triton-100, incubate at room temperature for 30 min), wash 3 times with PBS, 5 min each time;

[0065] S503: Secondary color development; add DAB solution containing 0.1% nickel ammonium sulfate, develop under a microscope for 1-10 minutes, positive signal is black; stop color development with tap water, rinse with running water for 5 minutes.

[0066] Further, S6: Seal the film and acquire the image;

[0067] Specifically, the tissue sections are air-dried, mounted with glycerol gelatin, and images are captured using a bright-field microscope. It should be noted that glycerol gelatin is solid at room temperature and should be melted in a 50°C water bath before use. Furthermore, it is essential to ensure that the slides do not dry out after hydration in step S103 and before development.

[0068] Example 2:

[0069] Example 2 provides a single-enzyme dual-substrate immunohistochemical double staining method for frozen tissue sections without primary antibody host species restrictions, specifically including the following steps:

[0070] S1: Pretreatment of frozen tissue sections;

[0071] Specifically, rinse the frozen tissue sections three times with distilled water for three minutes each time.

[0072] Further, step S2: Use Tris-EDTA antigen retrieval solution to perform antigen retrieval on the pretreated tissue sections;

[0073] S3: Perform the first staining using AEC chromogenic solution;

[0074] S4: Debonded primary and secondary antibodies were removed from sections immersed in a Tris-EDTA sodium citrate mixture by heating;

[0075] S5: Perform a second staining using DAB colorimetric solution containing 0.1% nickel ammonium sulfate;

[0076] S6: Seal the film and acquire the image.

[0077] The specific operations of steps S2 to S6 are exactly the same as those in Example 1.

[0078] Application Example 1:

[0079] Application Example 1: Double staining of astrocyte marker GFAP and microglia marker Iba-1 with catecholamine neuron marker tyrosine hydroxylase (TH).

[0080] This application example studies the substantia nigra pars compacta of brain tissue from an MPTP-induced PD mouse model fixed in 4% paraformaldehyde and embedded in paraffin. The specific experimental steps are as follows:

[0081] S1: Slice pretreatment;

[0082] Specifically, the tissue sections were divided into two groups, and each group underwent the following procedures:

[0083] Baking: Baking at 65℃ for 1 hour;

[0084] Dewaxing: Dewaxing with xylene 3 times, 10 minutes each time;

[0085] Hydration: Soak in 100% alcohol, 95% alcohol, 85% alcohol and 75% alcohol for 5 minutes in sequence, then rinse with running water for 5 minutes, rinse once with distilled water for 3 minutes.

[0086] S2: Antigen retrieval;

[0087] First, boil the Tris-EDTA antigen retrieval solution on high heat. Then, immerse the pretreated slides from step S1 in the antigen retrieval solution and continue microwave heating. After boiling, reduce the heat to medium and maintain heating for 10 minutes. Then, turn off the heat.

[0088] S3: Inactivates endogenous peroxidase and blocks non-specific binding sites;

[0089] Use a drawing tool to circle the tissue section; add 3% hydrogen peroxide and incubate at room temperature for 20 minutes.

[0090] Blocking: Add 10% goat serum to block, and incubate at room temperature for 20 minutes.

[0091] S4: First staining;

[0092] Incubate with the first primary antibody: Discard the serum, do not wash, add the first primary antibody: rabbit-derived anti-TH (Proteintech, #25859-1-AP, 1:1000), the primary antibody dilution is 10% goat serum, incubate at 37°C for 1 h; wash 3 times with PBS, 5 min each time;

[0093] Incubation with the first HRP-labeled secondary antibody: Add the first secondary antibody, HRP-labeled goat anti-rabbit IgG (Abcam, #ab6721, 1:1000), with 10% goat serum as the dilution buffer, and incubate at room temperature for 30 min; wash 3 times with PBS, 5 min each time;

[0094] First color development: Add AEC working solution (prepared fresh for use), develop color for 10 minutes, positive signal is red; stop with tap water, rinse with running water for 5 minutes.

[0095] S5: Elute antibodies and HRP, and perform a second blocking step;

[0096] Elution of antibodies and HRP: Immerse the tissue sections after the first staining in the aforementioned Tris-EDTA sodium citrate mixture, heat in a microwave oven to boiling, then reduce to medium heat and maintain for 10 min to elute the bound primary and secondary antibodies; allow to cool naturally, rinse with running water for 5 min, rinse once with distilled water for 3 min.

[0097] Second round of blocking: Add 10% goat serum for blocking and incubate at room temperature for 20 minutes.

[0098] S6: Second staining;

[0099] Incubate with the second primary antibody: Discard the serum, do not wash, add the second primary antibody to the first group of slides, rabbit-derived Anti-GFAP (Sigma-Aldrich, HPA056030, 1:500), add the first primary antibody to the second group of slides: rabbit-derived Anti-Iba-1 (CST, #17198, 1:300), the second round of primary antibody is diluted with 10% goat serum containing 0.1% Triton-100, incubate at room temperature for 1.5 h, wash with PBS 3 times, 5 min each time;

[0100] Incubate with the second HRP-labeled secondary antibody: Add the second secondary antibody, HRP-labeled goat anti-rabbit IgG (Abcam, #ab6721, 1:1000), diluted with 10% goat serum containing 0.1% Triton-100, and incubate at room temperature for 30 min; wash three times with PBS, 5 min each time;

[0101] Second color development: Add DAB solution containing 0.1% nickel ammonium sulfate, develop color under a microscope for 3 minutes, positive signal is black; stop with tap water, rinse with running water for 5 minutes.

[0102] S7: Seal the film and acquire the image;

[0103] The tissue sections were air-dried, mounted with glycerol gelatin, and images were captured using a bright-field microscope.

[0104] The attached image shows the double staining results obtained using a bright-field microscope in this application example. Figure 1 As shown, SNc represents *substantia nigrapars compacta*, the compact portion of the nitric acid. (From the appendix...) Figure 1 It can be seen from this:

[0105] A: In the first group of slices, the TH immunoreaction is red (AEC), representing dopaminergic neurons, and the GFAP immunoreaction is black (DAB nickel ion enhanced), representing astrocytes. The red and black are clearly distinguishable and there is no cross-reaction.

[0106] B: In the second group of slices, the TH immunoreaction is red (AEC), representing dopaminergic neurons, and the Iba-1 immunoreaction is black (DAB nickel ion enhanced), representing microglia. The red and black are clearly distinguishable and there is no cross-reaction.

[0107] Application Example 2:

[0108] Application example 2 is double staining of p-α-synuclein (S129) with TH, a marker of catecholamine neurons.

[0109] This application example studies the locus coeruleus of the pons from SNCA-A53T mutant transgenic mouse brain tissue fixed in 4% paraformaldehyde and embedded in paraffin. The specific experimental steps are as follows:

[0110] S1: Slice pretreatment;

[0111] Specifically, the tissue sections were divided into three groups, and each group was subjected to:

[0112] Baking: Baking at 65℃ for 1 hour;

[0113] Dewaxing: Dewaxing with xylene 3 times, 10 minutes each time;

[0114] Hydration: Soak in 100% alcohol, 95% alcohol, 85% alcohol and 75% alcohol for 5 minutes in sequence, then rinse with running water for 5 minutes, rinse once with distilled water for 3 minutes.

[0115] S2: Antigen retrieval;

[0116] First, boil the Tris-EDTA antigen retrieval solution on high heat. Then, immerse the pretreated slides from step S1 in the antigen retrieval solution and continue microwave heating. After boiling, reduce the heat to medium and maintain heating for 10 minutes. Then, turn off the heat.

[0117] S3: Inactivates endogenous peroxidase and blocks non-specific binding sites;

[0118] Use a drawing tool to circle the tissue section; add 3% hydrogen peroxide and incubate at room temperature for 20 minutes.

[0119] Blocking: Add 10% goat serum to block, and incubate at room temperature for 20 minutes.

[0120] S4: First staining;

[0121] Incubation with the first primary antibody: Discard the serum, do not wash, add the first primary antibody, rabbit-derived Anti-p-α-synuclein (S129) (Abcam, #ab51253, 1:300), the primary antibody dilution is 10% goat serum, incubate at 37°C for 1 h; wash 3 times with PBS, 5 min each time;

[0122] Incubation with the first HRP-labeled secondary antibody: Add the first secondary antibody, HRP-labeled goat anti-rabbit IgG (Abcam, #ab6721, 1:1000), with 10% goat serum as the dilution buffer, and incubate at room temperature for 30 min; wash 3 times with PBS, 5 min each time;

[0123] First stage of color development: DAB working solution (prepared fresh) was added to the first and second groups, and color development was carried out for 5 minutes. The positive signal was brownish-yellow. AEC working solution (prepared fresh) was added to the third group of sections, and color development was carried out for 30 minutes. The positive signal was red. The process was stopped with tap water and rinsed with running water for 5 minutes.

[0124] S5: Elute antibodies and HRP, and perform a second blocking step;

[0125] Elution of antibodies and HRP: Immerse tissue sections in the aforementioned Tris-EDTA sodium citrate mixture, heat in a microwave oven to boiling, then reduce to medium heat and maintain for 10 minutes to elute the bound primary and secondary antibodies; allow to cool naturally, rinse with running water for 5 minutes, rinse once with distilled water for 3 minutes.

[0126] Second round of blocking: Add 10% goat serum for blocking and incubate at room temperature for 20 minutes.

[0127] S6: Second staining;

[0128] Incubate with the second primary antibody: Discard the serum, do not wash, add the second primary antibody, rabbit-derived anti-TH (Proteintech, #25859-1-AP, 1:1000), diluted with 10% goat serum containing 0.1% Triton-100, incubate at room temperature for 1.5 h, wash 3 times with PBS for 5 min each time;

[0129] Incubate with the second HRP-labeled secondary antibody: Add the second secondary antibody, HRP-labeled goat anti-rabbit IgG (Abcam, #ab6721, 1:1000), diluted with 10% goat serum containing 0.1% Triton-100, and incubate at room temperature for 30 min; wash three times with PBS, 5 min each time;

[0130] Secondary staining: AEC staining solution was added to the first group of slides, and staining was performed under a microscope for 10 minutes. The positive signal was red. DAB solution containing 0.1% nickel ammonium sulfate was added to the second and third groups of slides, and staining was performed under a microscope for 3 minutes. The positive signal was black. The staining was stopped with tap water and rinsed with running water for 5 minutes.

[0131] S7: Seal the film and acquire the image;

[0132] The tissue sections were air-dried, mounted with glycerol gelatin, and images were captured using a bright-field microscope.

[0133] The bright-field microscope images of the three sets of slides are attached. Figure 2 As shown in A, B, and C, where LC stands for *locuscoeruleus*, the blue spot. From the appendix... Figure 2 It can be seen from this:

[0134] A: In the first group of slides, the S129 immunoreaction was brownish-yellow (DAB), and the TH immunoreaction was dark red (AEC). The contrast between the brownish-yellow and dark red colors was very low, making it difficult to distinguish the expression localization of the two.

[0135] B: In the second group of slides, the S129 immunoreaction was brownish-yellow (DAB), and the TH immunoreaction was black (DAB nickel ion enhanced). The contrast between the brownish-yellow and black colors was low, and there was a co-localization relationship between the two, but it was not easy to detect.

[0136] C: In the third group of slices, the S129 immunoreaction was red (AEC), and the TH immunoreaction was black (DAB nickel ion enhanced). The nuclei of TH-positive neurons (noradrenergic neurons) showed obvious S129 positivity. The red and black colors were clearly distinct, with no cross-reaction, and there was obvious co-localization between the two.

[0137] Application Example 3:

[0138] Application example 3 is double staining of aromatic amino acid decarboxylase AADC with oligodendrocyte marker Olig2.

[0139] This application example studies the corpus callosum of wild-type C57BL / 6J mouse brain tissue fixed in 4% paraformaldehyde and embedded in paraffin. The specific experimental steps are as follows:

[0140] S1: Slice pretreatment;

[0141] Specifically, the tissue sections were divided into two groups, and each group underwent the following procedures:

[0142] Baking: Baking at 65℃ for 1 hour;

[0143] Dewaxing: Dewaxing with xylene 3 times, 10 minutes each time;

[0144] Hydration: Soak in 100% alcohol, 95% alcohol, 85% alcohol and 75% alcohol for 5 minutes in sequence, then rinse with running water for 5 minutes, rinse once with distilled water for 3 minutes.

[0145] S2: Antigen retrieval;

[0146] First, boil the Tris-EDTA antigen retrieval solution on high heat. Then, immerse the pretreated slides from step S1 in the antigen retrieval solution and continue microwave heating. After boiling, reduce the heat to medium and maintain heating for 10 minutes. Then, turn off the heat.

[0147] S3: Inactivates endogenous peroxidase and blocks non-specific binding sites;

[0148] Use a drawing tool to circle the tissue section; add 3% hydrogen peroxide and incubate at room temperature for 20 minutes.

[0149] Blocking: Add 10% goat serum to block, and incubate at room temperature for 20 minutes.

[0150] S4: First staining;

[0151] Incubate with the first primary antibody: Discard the serum, do not wash, add the first primary antibody, rabbit-derived anti-AADC (Proteintech, #10166-1-AP, 1:300), the primary antibody dilution is 10% goat serum, incubate at 37°C for 1 h; wash 3 times with PBS, 5 min each time;

[0152] Incubation with the first HRP-labeled secondary antibody: Add the first secondary antibody, HRP-labeled goat anti-rabbit IgG (Abcam, #ab6721, 1:1000), with 10% goat serum as the dilution buffer, and incubate at room temperature for 30 min; wash 3 times with PBS, 5 min each time;

[0153] First stage of color development: DAB working solution (prepared and used immediately) was added to the first group of slides, and color development was performed for 5 minutes. The positive signal was brownish-yellow. AEC working solution (prepared and used immediately) was added to the second group of slides, and color development was performed for 30 minutes. The positive signal was red. The process was stopped with tap water and rinsed with running water for 5 minutes.

[0154] S5: Elute antibodies and HRP, and perform a second blocking step;

[0155] Elution of antibodies and HRP: Immerse tissue sections in the aforementioned Tris-EDTA sodium citrate mixture, heat in a microwave oven to boiling, then reduce to medium heat and maintain for 10 minutes to elute the bound primary and secondary antibodies; allow to cool naturally, rinse with running water for 5 minutes, rinse once with distilled water for 3 minutes.

[0156] Second round of blocking: Add 10% goat serum for blocking, and incubate at 37°C for 10 min.

[0157] S6: Second staining;

[0158] Incubate with the second primary antibody: Discard the serum, do not wash, add the second primary antibody, rabbit-derived Anti-Olig2 (Abcam, #ab109186, 1:100), diluted with 10% goat serum containing 0.1% Triton-100, incubate at room temperature for 1.5 h, wash with PBS 3 times, 5 min each time;

[0159] Incubate with the second HRP-labeled secondary antibody: Add the second secondary antibody, HRP-labeled goat anti-rabbit IgG (Abcam, #ab6721, 1:1000), diluted with 10% goat serum containing 0.1% Triton-100, and incubate at room temperature for 30 min; wash three times with PBS, 5 min each time;

[0160] Secondary color development: Add AEC working solution (prepared fresh) to the first group of slides and develop under a microscope for 10 minutes; the positive signal is red. Add DAB solution containing 0.1% nickel ammonium sulfate to the second group of slides and develop for 3 minutes; the positive signal is black. Stop with tap water and rinse with running water for 5 minutes.

[0161] S7: Seal the film and acquire the image;

[0162] The tissue sections were air-dried, mounted with glycerol gelatin, and images were captured using a bright-field microscope.

[0163] Bright-field microscope images of the two sets of slides are attached. Figure 3 As shown in A and B, CC stands for Corpus callosum. (From the appendix...) Figure 3 It can be seen from this:

[0164] A: In the first group of slides, the AADC immunoreaction was brownish-yellow (DAB), and the Olig2 immunoreaction was red (AEC). The low contrast between the brownish-yellow and dark red colors makes it difficult to distinguish their localization relationship.

[0165] B: In the second set of sections, the AADC immunoreaction is red (AEC), and the Olig2 immunoreaction is black (DAB). The red and black colors are clearly distinct, with no cross-reaction. Olig2 (oligodendrocytes) and AADC show obvious co-localization. Bright-field visualization of the two co-localized antigens was achieved.

[0166] The foregoing has shown and described the basic principles, main features, and advantages of the present invention. Those skilled in the art should understand that the present invention is not limited to the above embodiments. The embodiments and descriptions in the specification are merely illustrative of the principles of the invention. Various changes and modifications can be made to the invention without departing from its spirit and scope, and all such changes and modifications fall within the scope of the present invention as claimed. The scope of protection of this invention is defined by the appended claims and their equivalents.

Claims

1. A single-enzyme dual-substrate immunohistochemical double staining method without primary antibody host species restrictions, characterized in that, Includes the following steps: S1: Tissue section pretreatment; S2: Use Tris-EDTA antigen retrieval solution to perform antigen retrieval on pretreated tissue sections; S3: Perform the first staining using AEC chromogenic solution; S4: Debonded primary and secondary antibodies were removed from sections immersed in a Tris-EDTA sodium citrate mixture by heating; S5: Perform a second staining using DAB colorimetric solution containing 0.1% nickel ammonium sulfate; S6: Seal the film and acquire the image.

2. The single-enzyme dual-substrate immunohistochemical double staining method without primary antibody host species restriction according to claim 1, characterized in that, The Tris-EDTA antigen retrieval solution described in step S2 contains 1 mM EDTANa2·2H2O and 10 mM Tris.

3. The single-enzyme dual-substrate immunohistochemical double staining method without primary antibody host species restriction according to claim 1, characterized in that, The antigen retrieval method described in step S2 includes the following steps: placing the Tris-EDTA antigen retrieval solution in a microwave oven and preheating it to boiling on high heat, then immersing the pretreated tissue slices in the Tris-EDTA antigen retrieval solution, continuing to heat it to boiling on high heat, immediately turning to medium heat and maintaining it for 10 minutes, and then allowing it to cool naturally to room temperature.

4. The single-enzyme dual-substrate immunohistochemical double staining method without primary antibody host species restriction according to claim 3, characterized in that, The first staining method in step S3 includes the following steps: S301: Incubate with the first primary antibody, then wash with PBS; the first primary antibody dilution is 10% secondary antibody homologous serum. S302: Incubate with the first HRP-labeled secondary antibody, then wash with PBS; the first secondary antibody dilution is 10% homologous serum. S303: First color development: Add AEC working solution, develop color for 10-30 minutes, positive signal is red, rinse with running water.

5. The single-enzyme dual-substrate immunohistochemical double staining method without primary antibody host species restriction according to claim 4, characterized in that, Step S4 includes the following steps: Immerse the tissue sections after AEC staining in a mixture of Tris-EDTA sodium citrate, heat to boiling on high in a microwave oven, immediately turn to medium heat and maintain for 10 minutes, then let cool naturally to room temperature, rinse with running water and then rinse with distilled water.

6. The single-enzyme dual-substrate immunohistochemical double staining method without primary antibody host species restriction according to claim 5, characterized in that, The preparation method of the Tris-EDTA sodium citrate mixture is as follows: add 2mM citric acid to 10mM trisodium citrate dihydrate to prepare sodium citrate working solution, and then mix Tris-EDTA antigen retrieval solution and sodium citrate working solution at a volume ratio of 2:

1.

7. The single-enzyme dual-substrate immunohistochemical double staining method without primary antibody host species restriction according to claim 5, characterized in that, The second staining operation described in step S5 includes the following steps: S501: Incubate with the second primary antibody, then wash with PBS; the second primary antibody dilution solution is 10% homologous serum containing 0.1% Triton-100 secondary antibody. S502: Incubate with the second HRP-labeled secondary antibody, then wash with PBS; the second secondary antibody dilution solution is 10% homologous serum containing 0.1% Triton-100. S503: Secondary color development; add DAB solution containing 0.1% nickel ammonium sulfate, develop under a microscope for 1-10 minutes, positive signal is black, rinse with running water.

8. The single-enzyme dual-substrate immunohistochemical double staining method without primary antibody host species restriction according to claim 7, characterized in that, In step S6, the slide is sealed with glycerin gelatin.

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