A tissue staining method based on antigen retrieval treatment combined with silver staining

Abstract

The application provides a tissue staining method based on antigen repair treatment combined with silver staining, comprising the following steps: a) high-temperature heat repair of a tissue section to be treated with an antigen repair solution, and washing with water after cooling to obtain a repaired tissue section; b) silver staining of the repaired tissue section obtained in step a), dehydration, and then mounting to obtain a stained tissue section. Compared with the prior art, the tissue staining method based on antigen repair treatment combined with silver staining provided by the application adopts specific process steps and conditions to realize overall good interaction: first, the brain tissue is subjected to antigen repair treatment, and then the silver staining technology is adopted, which significantly improves the signal-to-noise ratio of the senile plaque region, and ideal staining effect is obtained, which can effectively improve the detail visibility in pathological detection, help more accurately evaluate the morphology and distribution characteristics of the senile plaque, and has a wide application prospect.

Description

Technical Field

[0001] This invention belongs to the field of tissue staining technology, and more specifically, relates to a tissue staining method based on antigen retrieval treatment combined with silver staining. Background Technology

[0002] Senile plaques are one of the hallmark pathological features of senile dementia, especially Alzheimer's disease (AD). They are primarily composed of amyloid-beta (Aβ) deposits and commonly appear in the cerebral cortex and hippocampus. The formation of senile plaques is closely related to the development of neurodegenerative diseases; therefore, the ability to clearly detect and observe senile plaques in tissues is of great significance for diagnosing and studying the mechanisms of these diseases.

[0003] Currently, researchers employ various techniques to observe and identify senile plaques in brain tissue, commonly including immunohistochemical staining, fluorescent labeling, and silver staining. Silver staining is a classic method that effectively visualizes nerve fibers and neuronal synapses, particularly useful in the study of neurodegenerative diseases, where it helps to clearly observe senile plaques. However, traditional silver staining techniques have limitations, especially in detecting minute lesions such as senile plaques. The signal-to-noise ratio is low, making it difficult to identify some small or early-stage senile plaques. Secondly, in some poor-quality samples, senile plaques are difficult to stain clearly with silver, appearing as blurry or indistinct patches.

[0004] In addition, the following technical solutions are also disclosed in the prior art:

[0005] (1) Combined application of immunohistochemical staining and antigen retrieval: Many researchers have attempted to combine immunohistochemical techniques with antigen retrieval methods to improve the detectability of target proteins (such as amyloid Aβ) in tissues. For example, in Alzheimer's disease research, heat-induced antigen retrieval (HIER) was used to treat brain slices, followed by immunohistochemical staining with antibodies against Aβ. This method can effectively improve the binding efficiency of antibodies and antigens, enhance the staining signal, and thus improve the visualization of senile plaques.

[0006] (2) Combination of staining and imaging techniques: Some researchers have combined high-resolution imaging techniques, such as fluorescence microscopy, confocal microscopy, or super-resolution microscopy, which can enhance the contrast and resolution of stained images, thereby better revealing the subtle changes and details of age spots.

[0007] While these methods have improved the signal-to-noise ratio of senile spots to some extent, they still have some limitations. For example, traditional silver staining methods may not achieve sufficiently high sensitivity after processing, especially in the detection of certain senile spots, and the reproducibility and specificity of staining still need to be improved. Summary of the Invention

[0008] In view of this, the purpose of this invention is to provide a tissue staining method based on antigen retrieval treatment combined with silver staining. This composite method improves the staining effect, effectively enhances the visibility of details in pathological examination, and helps to more accurately assess the morphology and distribution characteristics of senile spots.

[0009] This invention provides a tissue staining method based on antigen retrieval treatment combined with silver staining, comprising the following steps:

[0010] a) The tissue sections to be processed were subjected to high-temperature thermal repair with antigen repair solution, and after cooling, they were washed with water to obtain the repaired tissue sections.

[0011] b) Silver stain the repaired tissue sections obtained in step a), dehydrate them, and mount them to obtain stained tissue sections.

[0012] Preferably, the tissue slice to be processed in step a) is brain tissue from a person with AD.

[0013] Preferably, the antigen repair solution in step a) is a sodium citrate repair solution; the sodium citrate repair solution is composed of a citric acid solution, a sodium citrate solution and water in a volume ratio of (5-10):(45-40):50.

[0014] Preferably, the concentration of the citric acid solution is 0.05M to 0.15M; the concentration of the sodium citrate solution is 0.05M to 0.15M.

[0015] Preferably, the high-temperature thermal repair process described in step a) specifically includes:

[0016] Pour the antigen retrieval solution into the dedicated slide box of the antigen retrieval pot, place the tissue slide to be processed in it, click start, and perform high-temperature heat retrieval at 110℃~130℃. The retrieval is complete when the antigen retrieval pot emits a beep. Open the air valve of the antigen retrieval pot to release the air. After the air is released, open the lid of the antigen retrieval pot and wait for the slide to cool to room temperature in the antigen retrieval solution.

[0017] Preferably, the cooling temperature in step a) is 15°C to 35°C; and the water washing time is 20 min to 40 min.

[0018] Preferably, the silver staining process described in step b) is as follows:

[0019] First, soak the repaired tissue sections in silver nitrate solution in a water bath at 35℃~40℃ for 10min~30min, then rinse with water at room temperature for 1min~10min; then soak in silver ammonia solution for 10min~30min; next, add 3~4 drops of ammonia water to 100mL of water using a 200µL pipette, and soak the sections in this dilute ammonia solution for 10min~30min; then add 3~10 drops of colorimetric solution using a 200µL pipette, and wait for color development; after color development, soak the sections in 1%~10% sodium thiosulfate solution for 1min~10min; finally, rinse with water for 1min~10min.

[0020] Preferably, the dehydration process in step b) specifically includes:

[0021] Soak in 70% alcohol, 80% alcohol, and 90% alcohol for 4 to 6 minutes each, then soak in two bottles of 100% alcohol for 9 to 11 minutes each, and finally soak in two bottles of xylene for 9 to 11 minutes each.

[0022] Preferably, the sealing process in step b) specifically involves:

[0023] Add neutral resin to the tissue section, cover with a coverslip, place in a fume hood and wait for the resin to dry. Then observe under a light microscope and store at room temperature.

[0024] Preferably, before performing high-temperature heat repair on the tissue sections to be processed using the antigen retrieval solution in step a), the procedure further includes:

[0025] The sections are dewaxed to water; the specific process of dewaxing the sections to water is as follows:

[0026] Soak the slices in two bottles of xylene for 9 to 11 minutes each, then in two bottles of 100% alcohol for 9 to 11 minutes each, then in 95% alcohol for 4 to 6 minutes, 90% alcohol for 4 to 6 minutes, 80% alcohol for 4 to 6 minutes, and 70% alcohol for 4 to 6 minutes, and finally in water for 4 to 6 minutes.

[0027] This invention provides a tissue staining method based on antigen retrieval treatment combined with silver staining, comprising the following steps: a) subjecting the tissue sections to be treated to high-temperature heat retrieval with antigen retrieval solution, followed by cooling and washing with water to obtain retrieval-treated tissue sections; b) silver staining the retrieval-treated tissue sections obtained in step a), followed by dehydration and mounting to obtain stained tissue sections. Compared with existing technologies, the tissue staining method based on antigen retrieval treatment combined with silver staining provided by this invention employs specific process steps and conditions to achieve better overall interaction: firstly, antigen retrieval treatment is performed on brain tissue, followed by silver staining technology, which significantly improves the signal-to-noise ratio of senile plaque areas, achieving a more ideal staining effect. This effectively improves the visibility of details in pathological examination, helps to more accurately assess the morphology and distribution characteristics of senile plaques, and has broad application prospects. Attached Figure Description

[0028] Figure 1 A flowchart of a tissue staining method based on antigen retrieval treatment combined with silver staining provided in an embodiment of the present invention;

[0029] Figures 2-4 These are light microscopic images of age spots observed on slices treated with different methods in an embodiment of the present invention. Detailed Implementation

[0030] The technical solution of the present invention will be clearly and completely described below with reference to the embodiments of the present invention. Obviously, the described embodiments are only some embodiments of the present invention, and not all embodiments. Based on the embodiments of the present invention, all other embodiments obtained by those of ordinary skill in the art without creative effort are within the scope of protection of the present invention.

[0031] This invention provides a tissue staining method based on antigen retrieval treatment combined with silver staining, comprising the following steps:

[0032] a) The tissue sections to be processed were subjected to high-temperature thermal repair with antigen repair solution, and after cooling, they were washed with water to obtain the repaired tissue sections.

[0033] b) Silver stain the repaired tissue sections obtained in step a), dehydrate them, and mount them to obtain stained tissue sections.

[0034] On the one hand, this invention can solve the problem of excessively strong staining signals in the background area during the staining process of existing silver staining techniques. By performing antigen repair on the tissue before silver staining, the staining background can be effectively reduced and the signal-to-noise ratio can be improved, thereby improving the detail visibility of pathological detection, especially the detection of senile spots.

[0035] On the other hand, the present invention can also solve the problem of poor silver staining effect caused by poor sample quality, especially poor staining effect of age spots, such as blurry age spots; by using the staining steps of the present invention, the influence of sample quality on staining results can be effectively reduced. In particular, for staining age spots, the staining steps of the present invention can make age spots clearer, thereby making it easier to visually distinguish the regions where each age spot is located, and can improve sample utilization and staining efficiency.

[0036] Based on this, the present invention provides a tissue staining method based on antigen retrieval treatment combined with silver staining. This composite method improves the staining effect and solves the problem of poor staining effect of senile spots caused by sample quality issues.

[0037] The present invention first performs high-temperature thermal repair on the tissue sections to be processed using an antigen repair solution, and then washes them with water after cooling to obtain the repaired tissue sections.

[0038] In this invention, the tissue slices to be processed are preferably brain tissue from a person suffering from Alzheimer's disease (AD). This invention does not have any particular restrictions on the source of the brain tissue from the person suffering from AD; brain banks well-known to those skilled in the art can be used.

[0039] In this invention, before performing high-temperature thermal repair on the tissue sections to be treated with antigen retrieval solution, it is preferable to further include:

[0040] Slice and dewax to water.

[0041] In this invention, the process of dewaxing the slices to water is preferably as follows:

[0042] Soak the slices in two bottles of xylene for 9 to 11 minutes each, then in two bottles of 100% alcohol for 9 to 11 minutes each, then in 95% alcohol for 4 to 6 minutes, 90% alcohol for 4 to 6 minutes, 80% alcohol for 4 to 6 minutes, 70% alcohol for 4 to 6 minutes, and finally in water for 4 to 6 minutes.

[0043] More preferably:

[0044] Soak the slices in two bottles of xylene for 10 minutes each, then in two bottles of 100% alcohol for 10 minutes each, then in 95% alcohol for 5 minutes, 90% alcohol for 5 minutes, 80% alcohol for 5 minutes, and 70% alcohol for 5 minutes in sequence, and finally in water for 5 minutes.

[0045] In this invention, all the steps of xylene → gradient alcohol → distilled water are carried out at room temperature, and there are no special limitations on this.

[0046] In this invention, the antigen repair solution is preferably a sodium citrate repair solution; the sodium citrate repair solution is preferably composed of a citric acid solution, a sodium citrate solution and water in a volume ratio of (5-10):(45-40):50, more preferably composed of a citric acid solution, a sodium citrate solution and water in a volume ratio of (7-8):(43-42):50.

[0047] In this invention, the concentration of the citric acid solution is preferably 0.05M to 0.15M, more preferably 0.1M; the concentration of the sodium citrate solution is preferably 0.05M to 0.15M, more preferably 0.1M. The solutions are obtained using preparation methods well-known to those skilled in the art, and this invention does not impose any special limitations on them.

[0048] This invention employs antigen retrieval, a chemical treatment method. In this invention, thermal retrieval (e.g., high-temperature steam) removes cross-linking agents from the sample, enhancing dye binding and labeling effects. By first performing antigen retrieval treatment in tissue and then combining it with silver staining, this invention significantly improves the signal-to-noise ratio of senile plaques and reveals more small or early-stage senile plaques. The antigen retrieval step effectively removes the impression of interfering points such as fixatives in the tissue, exposing more target proteins and enhancing the effect of subsequent silver staining, thereby greatly improving the visualization and detection sensitivity of senile plaques. Furthermore, the innovation of this invention lies in overcoming the problems of weak signal and high detection difficulty in existing methods by combining antigen retrieval and silver staining techniques, showing great application potential, especially in high-resolution observation and early diagnosis. Through this technology, researchers can more clearly identify and analyze key pathological features in neurodegenerative diseases, providing more effective tools and methods for the early diagnosis, mechanism research, and drug development of related diseases.

[0049] In this invention, the high-temperature thermal repair process is preferably as follows:

[0050] Pour the antigen retrieval solution into the dedicated slide box of the antigen retrieval pot, place the tissue slide to be processed in it, click start, and perform high-temperature heat retrieval at 110℃~130℃. The retrieval is complete when the antigen retrieval pot emits a beep. Open the air valve of the antigen retrieval pot to release the air. After the air is released, open the lid of the antigen retrieval pot and wait for the slide to cool to room temperature in the antigen retrieval solution.

[0051] In this invention, the cooling temperature is preferably 15℃~35℃, more preferably 20℃~30℃; the water washing time is preferably 20min~40min, more preferably 30min.

[0052] After obtaining the repaired tissue sections, the present invention silver-stained the repaired tissue sections, dehydrated them, and then mounted them to obtain stained tissue sections.

[0053] In this invention, the silver dyeing process is preferably as follows:

[0054] First, soak the repaired tissue sections in silver nitrate solution in a water bath at 35℃~40℃ for 10min~30min, then rinse with water at room temperature for 1min~10min; then soak in silver ammonia solution for 10min~30min; next, add 3~4 drops of ammonia water to 100mL of water using a 200µL pipette, and soak the sections in this dilute ammonia solution for 10min~30min; then add 3~10 drops of colorimetric solution using a 200µL pipette, and wait for color development; after color development, soak the sections in 1%~10% sodium thiosulfate solution for 1min~10min; finally, rinse with water for 1min~10min.

[0055] More preferably:

[0056] First, the repaired tissue sections were soaked in silver nitrate solution in a 37°C water bath for 20 minutes, and then rinsed with water at room temperature for 5 minutes (unless otherwise specified, all subsequent processes were performed at room temperature). Then, the sections were soaked in silver ammonia solution for 20 minutes. After that, 3-4 drops of ammonia water were added to 100 mL of water using a 200 μL pipette, and the sections were soaked in this dilute ammonia solution for 20 minutes. Then, 3-10 drops of chromogenic solution were added using a 200 μL pipette, and the sections were allowed to develop. After the chromogenic development was completed, the sections were soaked in 5% sodium thiosulfate solution for 5 minutes. Finally, the sections were rinsed with water for 5 minutes.

[0057] The present invention does not impose any special restrictions on the source of the liquids required for silver dyeing, such as silver nitrate solution and silver ammonia solution. They can be prepared by methods well known to those skilled in the art or purchased as commercial products.

[0058] In this invention, the dehydration process is preferably specifically as follows:

[0059] Soak in 70% alcohol, 80% alcohol, and 90% alcohol for 4 to 6 minutes each, then soak in two bottles of 100% alcohol for 9 to 11 minutes each, and finally soak in two bottles of xylene for 9 to 11 minutes each.

[0060] More preferably:

[0061] Soak in 70% alcohol, 80% alcohol and 90% alcohol for 5 minutes each, then soak in two bottles of 100% alcohol for 10 minutes each, and finally soak in two bottles of xylene for 10 minutes each.

[0062] In this invention, the dehydration process is preferably carried out at room temperature, as is well known to those skilled in the art.

[0063] In this invention, the sealing process is preferably as follows:

[0064] Add neutral resin to the tissue section, cover with a coverslip, place in a fume hood and wait for the resin to dry. Then observe under a light microscope and store at room temperature.

[0065] Existing silver staining techniques have the following drawbacks:

[0066] (1) During tissue staining, the staining signal in the background area is too strong, thus obscuring the details of the target area (such as age spots) that needs to be observed. This phenomenon can occur in many traditional staining techniques, especially for silver staining, which cannot effectively distinguish between the target area and the background signal in the staining step.

[0067] (2) Poor sample quality leads to poor staining results, especially in the senile spot area, such as blurred senile spots.

[0068] To address the shortcomings of existing silver staining techniques, this invention provides a silver staining method combined with antigen retrieval treatment, which has the following beneficial effects:

[0069] (1) Solving the problem of excessive background: By performing antigen repair treatment on the tissue before staining, the method of the present invention can effectively enhance the staining signal of the target area and reduce the background staining of non-target areas; this treatment can clearly remove or reduce the background signal, making the staining effect more accurate and the target area (such as senile spots) more prominently displayed; the purpose of antigen repair treatment is to restore antigen epitopes, optimize the selectivity in the staining process, thereby significantly improving the signal-to-noise ratio and enhancing the staining clarity of the target area.

[0070] (2) Solving sample quality problems and clear staining: By combining antigen retrieval treatment with silver staining technology, this invention can make up for the impact of poor sample quality; antigen retrieval treatment can expose the antigenic components in the tissue more completely, which helps the silver staining reagent to bind effectively and avoids unclear staining effect due to improper sample processing or quality problems; through this method, clearer and more detailed staining can be achieved in tissue samples of different quality, especially for senile spots, which can significantly improve their visibility.

[0071] This invention provides a tissue staining method based on antigen retrieval treatment combined with silver staining, comprising the following steps: a) subjecting the tissue sections to be treated to high-temperature heat retrieval with antigen retrieval solution, followed by cooling and washing with water to obtain retrieval-treated tissue sections; b) silver staining the retrieval-treated tissue sections obtained in step a), followed by dehydration and mounting to obtain stained tissue sections. Compared with existing technologies, the tissue staining method based on antigen retrieval treatment combined with silver staining provided by this invention employs specific process steps and conditions to achieve better overall interaction: firstly, antigen retrieval treatment is performed on brain tissue, followed by silver staining technology, which significantly improves the signal-to-noise ratio of senile plaque areas, achieving a more ideal staining effect. This effectively improves the visibility of details in pathological examination, helps to more accurately assess the morphology and distribution characteristics of senile plaques, and has broad application prospects.

[0072] To further illustrate the present invention, the following embodiments will be described in detail.

[0073] Example

[0074] See Figure 1 As shown, Figure 1 A flowchart of a tissue staining method based on antigen retrieval treatment combined with silver staining provided in this embodiment of the invention; specifically including the following steps:

[0075] (1) Dewaxing the sections to water (xylene → graded alcohol → distilled water, all steps are performed at room temperature):

[0076] The slices were soaked in two bottles of xylene (the two bottles were indistinguishable) for 10 minutes each, then in two bottles of 100% alcohol (the two bottles were indistinguishable) for 10 minutes each, then in 95% alcohol for 5 minutes, then in 90% alcohol for 5 minutes, then in 80% alcohol for 5 minutes, then in 70% alcohol for 5 minutes, and finally in ddH2O for 5 minutes.

[0077] The slides used in this experiment were all brain tissue from people with Alzheimer's disease (AD), and were obtained from two brain banks: ① National Health and Disease Human Brain Tissue Resource Center (Anhui Medical University) ② Netherlands Brain Bank.

[0078] (2) Pour the antigen repair solution (sodium citrate repair solution is used in this embodiment) into the special slide box of the antigen repair pot, put in the tissue slides, click start, and perform high temperature heat repair to 120°C. When the antigen repair pot makes a beep, the repair is over. Open the air valve of the antigen repair pot to release the air. After the air is released, open the lid of the antigen repair pot and wait for the slides to cool to room temperature in the antigen repair solution.

[0079] The sodium citrate repair solution formula:

[0080] A) 0.1M citric acid solution (Critrate Bufter):

[0081] a) Citric acid monohydrate 21.01g [0.1mol; M: 210.14; C6H8O7·H2O; 5949-29-1];

[0082] b) ddH2O 1000mL;

[0083] B) 0.1M sodium citrate solution:

[0084] a) 29.41 g of trisodium citrate dihydrate [M: 294.10; C6H5O7·2H2O; CAS: 6132-04-3];

[0085] b) 1000ml of ddH2O;

[0086] C) Sodium citrate repair solution (for antigen microwave repair) per 100mL:

[0087] 7 mL of 0.1 M citric acid solution; 43 mL of 0.1 M sodium citrate solution; 50 mL of ddH2O.

[0088] (3) After the slides are cooled to room temperature in the antigen retrieval solution, they are thoroughly washed with ddH2O for 30 min.

[0089] (4) Soak the slices in 20% silver nitrate solution for 20 minutes, then place them in a 37°C water bath.

[0090] Preparation of silver nitrate solution: Dissolve 6g of solid silver nitrate [CAS: 7761-88-8] in 30mL of ddH2O.

[0091] (5) Wash the slices with ddH2O for 5 min, then RT (room temperature).

[0092] (6) Soak the sections in silver ammonia solution for 20 min, then RT.

[0093] Preparation of silver ammonia solution: Add evaporated ammonia water dropwise to the silver nitrate solution prepared in step 4 (evaporated ammonia water: concentrated ammonia water evaporates for about 20 minutes at room temperature). Stop adding dropwise when the solution becomes clear and transparent again. This solution is the silver ammonia solution.

[0094] (7) Add 3-4 drops of ammonia water to 100 mL ddH2O using a 200 μL pipette, and soak the slice in the dilute ammonia solution for 20 min.

[0095] (8) Add an appropriate amount of color developing solution (3-10 drops with a 200 μL pipette), wait for color development, RT.

[0096] (9) After the color development is completed, soak the sections in 5% sodium thiosulfate for 5 minutes, then RT.

[0097] (10) Wash with ddH2O for 5 min, then RT.

[0098] (11) Dehydration and transparency: (gradient alcohol → xylene, RT) Soak in 70% alcohol → 80% alcohol → 90% alcohol for 5 minutes each → Soak in two bottles of 100% alcohol (no difference between the two bottles) for 10 minutes each → Soak in two bottles of xylene (no difference between the two bottles) for 10 minutes each.

[0099] (12) Neutral resin mounting: wet mounting (lower the fume hood glass to arm height for protection), drop a small amount of neutral resin [CAS: 96949-21-2] onto the tissue, cover with a coverslip, place in the fume hood and wait for the resin to dry. Observe under a light microscope and store at room temperature (see observation results for...). Figure 2 , Figure 3 , Figure 4 ).

[0100] Figures 2-4 By calculating the signal-to-noise ratio (SNR) of the same senile plaque on slices treated with different antigens, it can be found that the SNR of senile plaques on slices treated with antigen retrieval is relatively higher than that on untreated slices.

[0101] Experimental results show that this invention, by innovatively combining antigen retrieval treatment with silver staining technology, provides an effective solution to the problem of blurred staining caused by excessive background and poor sample quality in existing silver staining techniques. This improved staining method can significantly improve the visibility of details in pathological areas such as senile spots, providing more precise technical means for neurological research and pathological diagnosis.

[0102] The above description of the disclosed embodiments enables those skilled in the art to make or use the invention. Various modifications to these embodiments will be readily apparent to those skilled in the art, and the general principles defined herein may be implemented in other embodiments without departing from the spirit or scope of the invention. Therefore, the invention is not to be limited to the embodiments shown herein, but is to be accorded the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A method for staining a tissue based on antigen retrieval treatment combined with silver staining, characterized by, Comprising the following steps: a) slice deparaffinization to water of the tissue section to be treated, high-temperature heat repair with an antigen repair solution, washing with water after cooling to obtain a repaired tissue section; the tissue section to be treated is human brain tissue with AD; the antigen repair solution is a sodium citrate repair solution; the process of high-temperature heat repair is as follows: pour the antigen repair solution into the slice box special for antigen repair pot, put the tissue section to be treated, click start, and perform high-temperature heat repair to 110~130 DEG C, wait for the antigen repair pot to give a prompt sound, which is the end of repair, open the air valve of the antigen repair pot to release the air, open the cover of the antigen repair pot after the air release is completed, and wait for the slice to cool to room temperature in the antigen repair solution; b) silver staining of the repaired tissue section obtained in step a), dehydration and mounting to obtain a stained tissue section.

2. The method of tissue staining based on antigen retrieval processing combined with silver staining according to claim 1, characterized in that, In step a), the sodium citrate repair solution is composed of citric acid solution, sodium citrate solution and water in a volume ratio of (5~10): (45~40):

50.

3. The method of tissue staining based on antigen retrieval processing combined with silver staining according to claim 2, characterized in that, The concentration of the citric acid solution is 0.05M~0.15M; the concentration of the sodium citrate solution is 0.05M~0.15M.

4. The method of claim 1, wherein the antigen retrieval process-based tissue staining method is silver staining. In step a), the cooling temperature is 15~35 DEG C; the water washing time is 20~40 min.

5. The method of claim 1, wherein the antigen retrieval process-based tissue staining method is silver staining. In step b), the process of silver staining is as follows: First, immerse the repaired tissue section in silver nitrate solution under the condition of 35~40 DEG C water bath for 10~30 min, then wash with water at room temperature for 1~10 min; then immerse in silver ammine solution for 10~30 min; then, in 100 mL of water, 3~4 drops of ammonia water are added by 200 microliter pipette, and the slice is immersed in the ammonia water solution for 10~30 min; then 3~10 drops of color developing solution are added by 200 microliter pipette, and color development is waited; after color development, immerse the slice in 1%~10% sodium thiosulfate for 1~10 min; finally, wash with water for 1~10 min.

6. The method of claim 1, wherein the antigen retrieval process-based tissue staining method is silver staining. In step b), the process of dehydration is as follows: Immerse in 70% alcohol, 80% alcohol, 90% alcohol for 4~6 min respectively, then immerse in two bottles of 100% alcohol for 9~11 min respectively, and finally immerse in two bottles of xylene for 9~11 min respectively.

7. The method of claim 1, wherein the antigen retrieval process-based tissue staining method is silver staining. In step b), the process of mounting is as follows: Add neutral gum to the tissue section, cover with a cover glass, and place in a fume hood to dry the gum, then observe under a light microscope and store at room temperature.

8. The method of tissue staining based on antigen retrieval processing combined with silver staining according to any one of claims 1-7, characterized in that, In step a), the process of slice deparaffinization to water is as follows: Immerse the slice in two bottles of xylene for 9~11 min respectively, then immerse in two bottles of 100% alcohol for 9~11 min respectively, then immerse in 95% alcohol for 4~6 min, 90% alcohol for 4~6 min, 80% alcohol for 4~6 min, 70% alcohol for 4~6 min, and finally immerse in water for 4~6 min.

Images