Tissue staining method based on combination of antigen repair treatment and silver staining

Abstract

The invention provides a tissue staining method based on combination of antigen repair treatment and silver staining, which comprises the following steps: a) performing high-temperature thermal repair on a to-be-treated tissue slice by using an antigen repair liquid, cooling, and cleaning with water to obtain a repaired tissue slice; and b) performing silver staining on the repaired tissue slice obtained in the step a), dehydrating and sealing the slice to obtain the stained tissue slice. Compared with the prior art, the tissue staining method based on combination of antigen repair treatment and silver staining provided by the invention adopts specific process steps and conditions to realize relatively good overall interaction: firstly, the brain tissue is subjected to antigen repair treatment, and then the silver staining technology is adopted, so that the signal-to-noise ratio of an age pigment region is remarkably improved; compared with the prior art, an ideal staining effect is obtained, detail visibility in pathological detection can be effectively improved, the method helps to more accurately evaluate the morphology and distribution characteristics of the age pigment, and the method has a wide application prospect.

Description

Technical Field

[0001] The invention belongs to the technical field of tissue staining, and more specifically, relates to a tissue staining method based on antigen repair treatment combined with silver staining. Background Art

[0002] Senile plaques are one of the hallmark pathological features of senile dementia (especially Alzheimer's disease, AD). They are mainly composed of amyloid-beta (Aβ) deposits and often appear in the cortex and hippocampus of the brain. The formation of senile plaques is closely related to the occurrence of neurodegenerative diseases. Therefore, being able to clearly detect and observe senile plaques in tissues is of great significance for diagnosing and studying the mechanisms of such diseases.

[0003] Currently, researchers use various technical methods to observe and identify senile plaques in brain tissue, including immunohistochemical staining, fluorescent labeling, and silver staining. Silver staining is a classic staining method that can effectively visualize nerve fibers and neuronal synapses, especially when studying neurodegenerative diseases. Silver staining can help to clearly observe senile plaques. However, traditional silver staining technology has its limitations, especially in the detection of minor lesions such as senile plaques. The signal-to-noise ratio is not high, which makes some tiny or early senile plaques difficult to identify. Secondly, for some poor quality samples, the senile plaques on them are difficult to be clearly stained with silver, such as appearing blurred.

[0004] In addition, the prior art also discloses the following technical solutions:

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

[0007] Although these methods have improved the signal-to-noise ratio of senile plaques to a certain extent, there are still some shortcomings. For example, the traditional silver staining method may not be able to obtain a high enough sensitivity after processing, especially in the detection of some senile plaques, and the repeatability and specificity of staining still need to be improved. Summary of the invention

[0008] In view of this, the purpose of the present invention is to provide a tissue staining method based on antigen retrieval treatment combined with silver staining. Through this composite method, the staining effect can be improved, the visibility of details in pathological detection can be effectively improved, and the morphology and distribution characteristics of senile plaques can be more accurately evaluated.

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

[0010] a) performing high-temperature heat repair on the tissue slice to be processed using an antigen repair solution, cooling the slice and washing it with water to obtain a repaired tissue slice;

[0011] b) silver staining the repaired tissue sections obtained in step a), dehydrating and sealing the sections to obtain stained tissue sections.

[0012] Preferably, the tissue section to be processed in step a) is human brain tissue suffering from AD.

[0013] Preferably, the antigen repair solution in step a) is sodium citrate repair solution; 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.

[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 in step a) is specifically as follows:

[0016] Pour the antigen repair solution into the special slice box of the antigen repair pot, put in the tissue slices to be processed, click start, and perform high-temperature heat repair to 110℃~130℃. When the antigen repair pot emits a prompt sound, the repair is completed. Open the air valve of the antigen repair pot to deflate. After deflation, open the lid of the antigen repair pot and wait for the slices to cool to room temperature in the antigen repair solution.

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

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

[0019] First, the repaired tissue sections are soaked in a silver nitrate solution at 35°C to 40°C water bath conditions for 10min to 30min, and then washed with water at room temperature for 1min to 10min; then soaked in a silver ammonia solution for 10min to 30min; then, 3 to 4 drops of ammonia water are dripped into 100mL of water with a 200-microliter pipette, and the sections are soaked in the dilute ammonia solution for 10min to 30min; then 3 to 10 drops of color developing solution are dripped with a 200-microliter pipette, and color development is waited; after color development is completed, the sections are soaked in 1% to 10% sodium thiosulfate for 1min to 10min; finally, washed with water for 1min to 10min.

[0020] Preferably, the dehydration process in step b) is specifically as follows:

[0021] Soak in 70% alcohol, 80% alcohol, and 90% alcohol for 4 to 6 minutes respectively, 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 respectively.

[0022] Preferably, the sealing process in step b) is specifically as follows:

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

[0024] Preferably, before the step a) of subjecting the tissue section to be processed to high temperature heat repair using an antigen repair solution, the step further includes:

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

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

[0027] The present invention provides a tissue staining method based on antigen repair treatment combined with silver staining, comprising the following steps: a) subjecting the tissue slice to be treated to high temperature heat repair with antigen repair solution, cooling and washing with water to obtain the repaired tissue slice; b) subjecting the repaired tissue slice obtained in step a) to silver staining, dehydrating and sealing to obtain the stained tissue slice. Compared with the prior art, the tissue staining method based on antigen repair treatment combined with silver staining provided by the present invention adopts specific process steps and conditions to achieve overall better interaction: firstly, the brain tissue is subjected to antigen repair treatment, and then silver staining technology is adopted, which significantly improves the signal-to-noise ratio of the senile plaque area, obtains a relatively ideal staining effect, can effectively improve the visibility of details in pathological detection, and helps to more accurately evaluate the morphology and distribution characteristics of senile plaques, and has broad application prospects. BRIEF DESCRIPTION OF THE DRAWINGS

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

[0029] Figures 2 to 4 These are photos of light microscopic observation results of senile plaques on slices with different treatments in the examples of the present invention. DETAILED DESCRIPTION

[0030] The technical solution of the present invention will be clearly and completely described below in conjunction with the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

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

[0032] a) performing high-temperature heat repair on the tissue slice to be processed using an antigen repair solution, cooling the slice and washing it with water to obtain a repaired tissue slice;

[0033] b) silver staining the repaired tissue sections obtained in step a), dehydrating and sealing the sections to obtain stained tissue sections.

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

[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 blurred age spots; using the staining steps of the present invention, the influence of sample quality on the staining results can be effectively reduced, especially for age spot staining, the staining steps of the present invention can make the age spots clearer, thereby visually making it easier to distinguish the areas where the age spots are located, and can improve sample utilization and staining efficiency.

[0036] On this basis, the present invention provides a tissue staining method based on antigen repair treatment combined with silver staining, which improves the staining effect and solves the problem of poor age spot staining effect caused by sample quality problems.

[0037] The present invention firstly uses an antigen repair solution to perform high-temperature heat repair on the tissue slice to be processed, and then washes it with water after cooling to obtain the repaired tissue slice.

[0038] In the present invention, the tissue slice to be processed is preferably human brain tissue suffering from AD. The present invention has no particular limitation on the source of the human brain tissue suffering from AD, and a brain bank well known to those skilled in the art can be used.

[0039] In the present invention, before the tissue sections to be processed are subjected to high temperature heat repair using an antigen repair solution, the process preferably further comprises:

[0040] Dewax the sections into water.

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

[0042] Soak the slices in two bottles of xylene for 9 to 11 minutes, then in two bottles of 100% alcohol for 9 to 11 minutes, 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 min each, then in two bottles of 100% alcohol for 10 min each, then in 95% alcohol for 5 min, 90% alcohol for 5 min, 80% alcohol for 5 min, 70% alcohol for 5 min, and finally in water for 5 min.

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

[0046] In the present 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, and 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 the present 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 above solutions are obtained by a preparation method well known to those skilled in the art, and the present invention has no particular limitation thereto.

[0048] The antigen repair used in the present invention is a chemical treatment method. In the present invention, the cross-linking agent in the sample is removed by heat repair (such as high-temperature steam) in the antigen repair, thereby enhancing the binding and labeling effect of the dye. The present invention can significantly improve the signal-to-noise ratio of senile plaques and reveal more tiny or early senile plaques by first performing antigen repair treatment in the tissue and then combining silver staining technology; the antigen repair step effectively removes the impression of interference points such as fixatives in the tissue, exposes more target proteins, and enhances the effect of subsequent silver staining, thereby greatly improving the visualization effect and detection sensitivity of senile plaques. On this basis, the innovation of the present invention is that by combining the use of antigen repair and silver staining technology, the problems of weak signals and difficult detection in existing methods are overcome, especially in high-resolution observation and early diagnosis, showing great application potential; through this technology, researchers can more clearly identify and analyze the key pathological features in neurodegenerative diseases, and provide more effective tools and methods for early diagnosis, mechanism research and drug development of related diseases.

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

[0050] Pour the antigen repair solution into the special slice box of the antigen repair pot, put in the tissue slices to be processed, click start, and perform high-temperature heat repair to 110℃~130℃. When the antigen repair pot emits a prompt sound, the repair is completed. Open the air valve of the antigen repair pot to deflate. After deflation, open the lid of the antigen repair pot and wait for the slices to cool to room temperature in the antigen repair solution.

[0051] In the present invention, the cooling temperature is preferably 15°C to 35°C, more preferably 20°C to 30°C; the washing time with water is preferably 20min to 40min, more preferably 30min.

[0052] After obtaining the repaired tissue slices, the present invention performs silver staining on the repaired tissue slices, dehydrates and seals the slices to obtain stained tissue slices.

[0053] In the present invention, the silver staining process is preferably as follows:

[0054] First, the repaired tissue slices are soaked in a silver nitrate solution at 35°C to 40°C water bath for 10min to 30min, and then washed with water at room temperature for 1min to 10min; then soaked in a silver ammonia solution for 10min to 30min; then, 3 to 4 drops of ammonia water are dripped into 100mL of water with a 200 microliter pipette, and the slices are soaked in the dilute ammonia solution for 10min to 30min; then, 3 to 10 drops of color developing solution are dripped with a 200 microliter pipette, and color development is waited; after color development is completed, the slices are soaked in 1% to 10% sodium thiosulfate for 1min to 10min; finally, washed with water for 1min to 10min;

[0055] More preferably:

[0056] First, soak the repaired tissue sections in a silver nitrate solution in a 37°C water bath for 20 minutes, then wash with water at room temperature for 5 minutes (the subsequent process is at room temperature unless otherwise specified); then soak in a silver ammonia solution for 20 minutes; then, drop 3 to 4 drops of ammonia water into 100 mL of water with a 200-microliter pipette, and soak the sections in the dilute ammonia solution for 20 minutes; then drop 3 to 10 drops of color developing solution with a 200-microliter pipette, and wait for color development; after color development is completed, soak the sections in 5% sodium thiosulfate for 5 minutes; finally, wash with water for 5 minutes.

[0057] The present invention has no particular restriction on the source of the liquid required for silver staining, such as the silver nitrate solution and the silver ammonia solution, and the liquid can be prepared by a preparation method well known to those skilled in the art or purchased from a commercial product.

[0058] In the present 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 the present invention, the dehydration process is preferably carried out at room temperature well known to those skilled in the art.

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

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

[0065] The existing silver staining technology has the following disadvantages:

[0066] (1) During tissue staining, the staining signal in the background area is too strong, thus masking the details of the target area to be observed (such as senile plaques); this phenomenon may occur in many traditional staining techniques, especially for silver staining, which cannot effectively distinguish between the target area and the background signal during the staining step.

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

[0068] In view of the shortcomings of the above-mentioned existing silver staining technology, the present invention provides a silver staining method combined with antigen repair 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 the non-target area; this treatment can clearly remove or reduce the background signal, making the staining effect more accurate and the target area (such as age spots) more prominently displayed; the purpose of antigen repair treatment is to restore the antigen epitope and 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 the problem of sample quality and achieving clear staining: By combining antigen repair treatment with silver staining technology, the present invention can compensate for the impact of poor sample quality; antigen repair treatment can expose the antigenic components in the tissue more completely, which helps to effectively bind the silver staining reagent and avoid unclear staining effects caused by improper sample processing or quality problems; through this method, clearer and more detailed staining can be achieved in tissue samples of different qualities, especially for senile plaque areas, which can significantly improve their visibility.

[0071] The present invention provides a tissue staining method based on antigen repair treatment combined with silver staining, comprising the following steps: a) subjecting the tissue slice to be treated to high temperature heat repair with antigen repair solution, cooling and washing with water to obtain the repaired tissue slice; b) subjecting the repaired tissue slice obtained in step a) to silver staining, dehydrating and sealing to obtain the stained tissue slice. Compared with the prior art, the tissue staining method based on antigen repair treatment combined with silver staining provided by the present invention adopts specific process steps and conditions to achieve overall better interaction: firstly, the brain tissue is subjected to antigen repair treatment, and then silver staining technology is adopted, which significantly improves the signal-to-noise ratio of the senile plaque area, obtains a relatively ideal staining effect, can effectively improve the visibility of details in pathological detection, and helps to more accurately evaluate the morphology and distribution characteristics of senile plaques, and has broad application prospects.

[0072] In order to further illustrate the present invention, the following examples are provided for detailed description.

[0073] Example

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

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

[0076] The sections were soaked in two bottles of xylene (the two bottles were the same) for 10 min each → soaked in two bottles of 100% alcohol (the two bottles were the same) for 10 min each → soaked in 95% alcohol for 5 min → soaked in 90% alcohol for 5 min → soaked in 80% alcohol for 5 min → soaked in 70% alcohol for 5 min → soaked in ddH2O for 5 min.

[0077] The slices in this experiment were all brain tissues from people with AD, and they came from two brain banks: ①National Health and Disease Human Brain Tissue Resource Center (Anhui Medical University) and ②Netherlands Brain Bank.

[0078] (2) Pour the antigen repair solution (sodium citrate repair solution is used in this example) into the special slice box of the antigen repair pot, put the tissue slices in, click start, and perform high-temperature heat repair to 120°C. When the antigen repair pot emits a prompt sound, the repair is completed. Open the air valve of the antigen repair pot to release air. After the release is completed, open the lid of the antigen repair pot and wait for the slices 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 21.01 g [0.1 mol; M: 210.14; C6H8O7·H2O; 5949-29-1];

[0082] b) ddH2O 1000mL;

[0083] B) 0.1M sodium citrate solution:

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

[0085] b) ddH2O 1000ml;

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

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

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

[0089] (4) Soak the sections in 20% silver nitrate solution for 20 min in a 37°C water bath.

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

[0091] (5) Wash the sections with ddH2O for 5 min at RT.

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

[0093] Preparation of silver ammonia solution: Add volatilized ammonia water (volatilized ammonia water: concentrated ammonia water evaporates for about 20 minutes at room temperature) to the silver nitrate solution prepared in step 4, and add until the solution becomes transparent and clear again, then stop adding. This solution is the silver ammonia solution.

[0094] (7) Use a 200 μL pipette to add 3 to 4 drops of aqueous ammonia to 100 mL of ddH2O. Soak the sections in the diluted aqueous ammonia solution for 20 min at RT.

[0095] (8) Add an appropriate amount of color developing solution (3 to 10 drops using a 200 μL pipette) and wait for color development at RT.

[0096] (9) After color development, soak the sections in 5% sodium thiosulfate for 5 min at RT.

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

[0098] (11) Dehydration and transparency: (gradient alcohol → xylene, RT) 70% alcohol → 80% alcohol → 90% alcohol, soak for 5 min each → two bottles of 100% alcohol (the two bottles are the same) soak for 10 min each → two bottles of xylene (the two bottles are the same) soak for 10 min each.

[0099] (12) Neutral gum sealing: Wet sealing (lower the fume hood glass to arm height for protection), drop a small amount of neutral gum [CAS: 96949-21-2] on the tissue, cover with a coverslip, place in the fume hood and wait for the gum to dry. It can be observed under a light microscope and stored at room temperature (for observation results, see Figure 2 , Figure 3 , Figure 4 ).

[0100] Figures 2 to 4 : By calculating the signal-to-noise ratio of the same senile plaque on differently treated slices, it can be found that the signal-to-noise ratio of senile plaques on slices treated with antigen retrieval is higher than that on slices without treatment.

[0101] The experimental results show that the present invention provides an effective solution to the problem of blurred staining caused by excessive background and poor sample quality in existing silver staining technology by innovatively combining antigen repair treatment with silver staining technology; through this improved staining method, the visibility of details of pathological areas such as senile plaques can be significantly improved, providing more accurate technical means for neurological research and pathological diagnosis.

[0102] The above description of the disclosed embodiments enables those skilled in the art to implement or use the present invention. Various modifications to these embodiments will be 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 present invention. Therefore, the present invention will not be limited to the embodiments shown herein, but rather to the widest scope consistent with the principles and novel features disclosed herein.

Claims

1. A tissue staining method based on antigen retrieval treatment combined with silver staining, characterized in that: The following steps are involved: a) performing high-temperature heat repair on the tissue slice to be processed using an antigen repair solution, cooling the slice and washing it with water to obtain a repaired tissue slice; b) silver staining the repaired tissue sections obtained in step a), dehydrating and sealing the sections to obtain stained tissue sections.

2. The tissue staining method based on antigen retrieval treatment combined with silver staining according to claim 1, characterized in that: The tissue slice to be processed in step a) is human brain tissue suffering from AD.

3. The tissue staining method based on antigen retrieval treatment combined with silver staining according to claim 1, characterized in that: The antigen repair solution in step a) is sodium citrate repair solution; 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.

4. The tissue staining method based on antigen retrieval treatment combined with silver staining according to claim 3, characterized in that: 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.

5. The tissue staining method based on antigen retrieval treatment combined with silver staining according to claim 1, characterized in that: The process of high temperature thermal repair in step a) is specifically as follows: Pour the antigen repair solution into the special slice box of the antigen repair pot, put in the tissue slices to be processed, click start, and perform high-temperature heat repair to 110℃~130℃. When the antigen repair pot emits a prompt sound, the repair is completed. Open the air valve of the antigen repair pot to deflate. After deflation, open the lid of the antigen repair pot and wait for the slices to cool to room temperature in the antigen repair solution.

6. The tissue staining method based on antigen retrieval treatment combined with silver staining according to claim 1, characterized in that: The cooling temperature in step a) is 15° C. to 35° C.; the washing time with water is 20 min to 40 min.

7. The tissue staining method based on antigen retrieval treatment combined with silver staining according to claim 1, characterized in that: The silver staining process in step b) is specifically as follows: First, the repaired tissue sections are soaked in a silver nitrate solution at 35°C to 40°C water bath conditions for 10min to 30min, and then washed with water at room temperature for 1min to 10min; then soaked in a silver ammonia solution for 10min to 30min; then, 3 to 4 drops of ammonia water are dripped into 100mL of water with a 200-microliter pipette, and the sections are soaked in the dilute ammonia solution for 10min to 30min; then 3 to 10 drops of color developing solution are dripped with a 200-microliter pipette, and color development is waited; after color development is completed, the sections are soaked in 1% to 10% sodium thiosulfate for 1min to 10min; finally, washed with water for 1min to 10min.

8. The tissue staining method based on antigen retrieval treatment combined with silver staining according to claim 1, characterized in that: The dehydration process in step b) is specifically as follows: Soak in 70% alcohol, 80% alcohol, and 90% alcohol for 4 to 6 minutes respectively, 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 respectively.

9. The tissue staining method based on antigen retrieval treatment combined with silver staining according to claim 1, characterized in that: The process of sealing the slide in step b) is specifically as follows: Add neutral gum to the tissue section, cover with a coverslip, and place in a fume hood to wait for the gum to dry. Then, observe under a light microscope and store at room temperature.

10. The tissue staining method based on antigen retrieval treatment combined with silver staining according to any one of claims 1 to 9, characterized in that: Before the tissue sections to be processed are subjected to high temperature heat repair using an antigen repair solution in step a), the method further comprises: The sections are dewaxed to water; the process of dewaxing the sections to water is specifically as follows: Soak the slices in two bottles of xylene for 9 min to 11 min respectively, then soak them in two bottles of 100% alcohol for 9 min to 11 min respectively, then soak them in 95% alcohol for 4 min to 6 min, 90% alcohol for 4 min to 6 min, 80% alcohol for 4 min to 6 min, 70% alcohol for 4 min to 6 min, and finally soak them in water for 4 min to 6 min.

Images