Dyeing kit and dyeing method for co-localization evaluation of pathology of enteritis model

Abstract

The invention relates to a dyeing kit and a dyeing method for colocalization evaluation of enteritis model pathology. The staining kit comprises the following components: a hematoxylin staining solution, an alcian blue staining solution, a periodic acid solution, a Schiff reagent, a dewaxing antigen repair solution, a peroxidase inhibitor, a direct-use primary antibody CD4, a non-specific antigen confining liquid, a direct-use secondary antibody HRP-goat anti-rabbit IgG polymer, a DAB color developing agent, a phosphate buffer solution, a Tris buffer salt solution and a phosphate buffer solution detergent. And a transparent agent. According to the method, histological pathological characteristics of an enteritis model and a scoring system thereof are ingeniously fused, multiple staining which needs to be completed by three slices traditionally can be integrated to one slice by adopting a single slice, complex steps of multiple staining methods in the early-stage slice preparation process are simplified and accelerated, time consumption is reduced, it is ensured that the slices can be completed more quickly, and the efficiency is improved. Therefore, the next analysis stage is timely entered.

Description

Technical Field

[0001] The invention belongs to the technical field of biological detection, and particularly relates to a staining kit and a staining method for pathological co-localization evaluation of an enteritis model. Background Art

[0002] Enteritis model is an important tool for studying enteritis diseases, especially ulcerative colitis (UC) model is the most widely used. The main manifestations of ulcerative colitis include diarrhea, mucoid stool, mucosal ulcer, intestinal wall thickening, fibrotic granuloma, angiogenesis, edema, inflammatory infiltration, goblet cell and secretory cell reduction or abnormal secretion affecting digestive flora disorder, etc. The effect and toxicity of related drug development need to be tested on related symptoms to know the drug effect and toxicological changes, so gene knockout or chemical induction is performed on animals to achieve histological changes in enteritis diseases.

[0003] The current method for studying the histological changes in enteritis is to prepare slides after the animal experiment, and then perform HE staining, immunohistochemistry staining, and special staining on sections of the same tissue at different depths for structural analysis and scoring evaluation.

[0004] The disadvantages of the existing technology are: (1) The process of preparing multiple slices in the early stage, including slicing, staining, scanning and quality control, is time-consuming (more than three days), which is not only tedious and laborious, but also a huge waste of time and cost for both the R&D client and the testing party; (2) During the tissue chemical staining stage, slice detachment is a common problem, which will lead to poor analysis results of multiple slices at the common positioning level. When cells are not at the same level, it will become more difficult to analyze and summarize the effects of drugs or devices, and the results will be highly biased; (3) The currently used methods are numerous and complex, and the conditions of use between different institutions are not consistent, resulting in the inability to completely replicate the same staining results, which further increases the uncertainty and difficulty of evaluation; (4) There are many quality differences in the R&D evaluation of a single drug in the industry. The large difference in results not only leads to huge financial investment by the R&D party, but also is not conducive to the improvement of drug R&D technology and mature market launch, becoming an important factor restricting the drug R&D process. Summary of the invention

[0005] The purpose of the present invention is to provide a staining kit and a staining method for pathological co-localization evaluation of enteritis models in order to solve the above problems.

[0006] The present invention achieves the above-mentioned purpose through the following technical solutions:

[0007] The invention provides a staining kit for pathological co-localization evaluation of an enteritis model. The staining kit comprises the following components: hematoxylin staining solution, alcian blue staining solution, periodic acid solution, Schiff reagent, dewaxing antigen repair solution (PH9.0), peroxidase inhibitor, direct-use primary antibody CD4, non-specific antigen blocking solution, direct-use secondary antibody HRP-goat anti-rabbit IgG polymer, DAB color developer, phosphate buffered saline (PBS), Tris buffered saline solution (PH7.6), phosphate buffered saline detergent, and a transparent agent.

[0008] As a further optimization scheme of the present invention, the Alcian blue staining solution is a 1% Alcian blue staining solution, and the method for obtaining the 1% Alcian blue staining solution is to add 1g of Alcian blue 8GX powder to 3ml of acetic acid and 97ml of distilled water, fully dissolve it, and let it stand for 1 hour to obtain it.

[0009] As a further optimization scheme of the present invention, the method for obtaining the periodic acid solution is to add 0.5 g of periodic acid to 100 ml of water and let it stand for 30 minutes.

[0010] As a further optimization scheme of the present invention, the Schiff reagent is prepared from 1g of basic fuchsin, 20ml of 1mol / L hydrochloric acid, 1.5g of sodium metabisulfite, 200ml of distilled water, and 2g of activated carbon;

[0011] The Schiff reagent is obtained by dissolving 1 g of basic fuchsin in 200 ml of distilled water at 80° C., heating and boiling for a while, and fully stirring, filtering when cooled to 50° C., adding 20 ml of 1 mol / L hydrochloric acid, and adding 1.5 g of sodium metabisulfite when cooled to 25° C., sealing and placing in a dark place at room temperature for 24 hours, at which time the solution is light khaki or light red, then adding 2 g of activated carbon powder, fully shaking the solution, and filtering after standing for 2 hours to obtain a colorless, clear and transparent solution.

[0012] As a further optimized solution of the present invention, the peroxidase inhibitor is prepared by preparing 10 parts of 30% hydrogen peroxide by mass and 90 parts of phosphate buffer.

[0013] As a further optimization scheme of the present invention, the direct-use primary antibody CD4 is prepared from 1 part of CD4 primary antibody, 1 part of bovine serum albumin, 0.1 part of sodium azide solution, and 97.9 parts of phosphate buffer.

[0014] As a further optimized solution of the present invention, the non-specific antigen blocking solution is prepared from 50 parts of goat serum, 49.9 parts of glycerol, and 0.1 parts of sodium azide solution.

[0015] As a further optimization scheme of the present invention, the phosphate buffer is prepared from 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, and 800 ml of deionized pure water.

[0016] As a further optimization scheme of the present invention, the pH of the Tris buffered saline solution is 7.6, and the Tris buffered saline solution is prepared from 6.05 g of tris(hydroxymethyl)aminomethane, 8.76 g of sodium chloride, and 800 ml of deionized pure water.

[0017] As a further optimized solution of the present invention, the phosphate buffer detergent is prepared from 99.5 parts of phosphate buffer and 200.5 parts of polysorbate.

[0018] The present invention also provides a dyeing method based on the above dyeing kit, the specific steps are:

[0019] 1) Preparation of paraffin sections of animal tissues;

[0020] 2) Dewaxing and antigen repair: preheat the vacuum drying oven to 93°C in advance, and use a container to pour 250 ml of dewaxing antigen repair solution (PH9.0) to soak the slices obtained in step 1) for 5 minutes; release the pressure, take out the slices, and soak and wash them in 70°C phosphate buffer for 3 times / 5 minutes each time, and wash them in room temperature Tris buffer solution for 3 times / 5 minutes each time;

[0021] 3) Peroxidase blocking: the slices treated in step 2) are blocked by dripping or soaking with a peroxidase inhibitor at room temperature for 30 minutes, the peroxidase inhibitor is removed and the slices are soaked in a phosphate buffer detergent 3 times / 5 minutes each time;

[0022] 4) Primary antibody labeling: the slices treated in step 3) were incubated with direct-use primary antibody CD4 at room temperature for 1 hour, and then soaked in phosphate buffer detergent 3 times / 5 minutes each time;

[0023] 5) The slices treated in step 4) were incubated in non-specific antigen blocking solution at room temperature for 30 minutes, and then soaked in phosphate buffer detergent 3 times / 5 minutes each time;

[0024] 6) Secondary antibody incubation: the slices treated in step 5) were incubated with direct secondary antibody HRP-goat anti-rabbit IgG polymer at room temperature for 15 minutes, and then soaked in phosphate buffer detergent 3 times / 5 minutes each time;

[0025] 7) DAB color development: the slices treated in step 6) were incubated with DAB color developer for 5 min at room temperature, and then soaked in phosphate buffer detergent once for 5 min each time;

[0026] 8) Alcian blue staining: the slices treated in step 7) are stained with Alcian blue staining solution at room temperature for 5 minutes, and then washed with pure water until no excess blue is removed;

[0027] 9) Glycogen oxidation: the slices treated in step 8) are immersed in periodic acid solution or dripped onto the slices and incubated at 2-8°C for 5 minutes, and then washed with deionized / pure water for 3 times;

[0028] 10) PAS staining: the slices treated in step 9) are immersed in Schiff reagent at 2-8°C for 5 minutes, and then rinsed with running water for 5-10 minutes until the liquid is colorless;

[0029] 11) Nuclear staining: the sections treated in step 10) were immersed in hematoxylin staining solution for 30 seconds, rinsed with running water for 2 minutes, and then rinsed with pure water for 3 times;

[0030] 12) Drying and sealing. The slices treated in step 11) are air-dried or dried at 60° C., and transparent agent is added to make them transparent, and then the slices are sealed with sealing glue.

[0031] The advantage of the staining method of the present invention is that it cleverly integrates the histological pathological characteristics of the enteritis model and its scoring system. The multiple staining that traditionally requires three slices to complete can be integrated into one slice using a single slice, and immunohistochemical staining of specific immune cell markers (brown), special staining of intestinal secretory cells (blue and purple-red), all cell nuclei in the tissue (light blue or bluish-white), cytoplasm and extracellular matrix staining (red or pink), and red blood cell staining (yellow) can be performed.

[0032] The beneficial effects of the present invention are:

[0033] 1) Compared with simply combining the three traditional staining methods for evaluation and analysis, the present invention has the advantage that the present invention can simplify and speed up the complex steps of multiple staining methods in the early stage of slide preparation and reduce the time consumption, ensuring that the slides can be completed more quickly, so as to enter the next analysis stage in time;

[0034] 2) In the process of slice preparation, the present invention ensures the high stability and integrity of the slices by using a rapid dewaxing and antigen repair method with vacuum negative pressure, effectively reduces the slice drop rate, and reduces the number of slices and the number of scans, which can significantly save time and reduce the cost of R&D enterprises accordingly;

[0035] 3) The present invention can effectively solve the problem of unstable results caused by co-localization analysis of multiple slices by implementing multiple staining technology on the same slice. This method not only improves the accuracy and reliability of the analysis, but also greatly saves the work of pathological diagnosis. There is no need to repeatedly switch different analysis slices or check multiple analysis results back and forth, so that pathological diagnosis and research can be carried out more efficiently. The application of this technology will undoubtedly greatly improve the efficiency and accuracy of pathological analysis;

[0036] 4) The present invention aims to accelerate the adoption process of many research institutions by establishing a unified pathological evaluation standard, effectively solving the problem of inconsistent drug evaluation results caused by differences in evaluation methods among various testing institutions, thereby promoting a significant improvement in economic benefits. BRIEF DESCRIPTION OF THE DRAWINGS

[0037] Figure 1 The section staining results (field of view 20X and field of view 40X) obtained by using the staining kit of Example 1 of the present invention to perform the staining experiment of Example 2 of the present invention;

[0038] Figure 2 This is a comparison chart of the section staining results of HE staining in the prior art and staining in the present invention;

[0039] Figure 3 It is a comparison chart of the section staining results of the immunohistochemical staining of the prior art and the staining of the present invention;

[0040] Figure 4 It is a comparison diagram of the section staining results of the prior art Alcian blue and PAS co-staining and the staining of the present invention;

[0041] Figure 5 It is a comparison diagram of the staining results of sections before and after dewaxing between the prior art and the present invention;

[0042] Figure 6 A comparison chart of the section staining results of the staining experiment operation of Example 2 of the present invention performed by different staff members (only the staff members are different);

[0043] Figure 7 This is a comparison chart of the section staining results of the staining experiment operation of Example 2 of the present invention performed by the same staff using staining kits with different storage times (only the storage time of the staining kit in Example 1 is different). DETAILED DESCRIPTION

[0044] The present application is further described in detail below in conjunction with the accompanying drawings. It is necessary to point out here that the following specific implementation methods are only used to further illustrate the present application and cannot be understood as limiting the scope of protection of the present application. Technical personnel in this field can make some non-essential improvements and adjustments to the present application based on the above application content.

[0045] 1. Materials

[0046] Unless otherwise specified, the methods used in the present invention are conventional methods known to those skilled in the art, and the reagents and other materials used are commercially available products unless otherwise specified.

[0047] 2. Methods

[0048] Example 1

[0049] A staining kit for pathological co-localization evaluation of an enteritis model comprises the following components: hematoxylin staining solution, alcian blue staining solution, periodic acid solution, Schiff reagent, dewaxing antigen repair solution (PH9.0), peroxidase inhibitor, direct-use primary antibody (CD4), nonspecific antigen blocking solution, direct-use secondary antibody HRP-goat anti-rabbit IgG polymer, DAB color developer, phosphate buffered saline (PBS), Tris buffered saline solution (PH7.6), phosphate buffer detergent, and a transparent agent.

[0050] Preferably, hematoxylin staining solution (manufacturer: Harbin Green Specimen Technology Development Co., Ltd., item number: GS-02).

[0051] Preferably, Alcian blue staining solution (1% Alcian blue staining solution): 1 g of Alcian blue 8GX powder is added into 3 ml of acetic acid and 97 ml of distilled water, fully dissolved, and allowed to stand for 1 hour before use.

[0052] Preferably, the periodic acid solution is prepared by adding 0.5 g periodic acid to 100 ml of water and letting it stand for 30 minutes before use.

[0053] Preferably, Schiff reagent: basic fuchsin 1g, 1mol / L hydrochloric acid 20ml, sodium metabisulfite 1.5g, distilled water 200ml, activated carbon 2g; first dissolve 1g basic fuchsin in 200ml distilled water at 80°C, then heat and boil for a while, and stir thoroughly, filter when cooled to 50°C, add 1mol / L hydrochloric acid 20ml, add 1.5g sodium metabisulfite when cooled to 25°C, seal and place in a dark place at room temperature for 24h, at which time the solution is light khaki or light red, then add 2g activated carbon powder, fully shake the solution, let it stand for 2h and then filter (at this time the solution is colorless, clear and transparent), and store in a brown bottle in a 4°C refrigerator.

[0054] Preferably, dewaxed antigen retrieval solution (PH 9.0) (manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., product number: DAS-0021).

[0055] Preferably, the peroxidase inhibitor includes 10 parts of 30% hydrogen peroxide and 90 parts of phosphate buffered saline (PBS).

[0056] Preferably, direct-use primary antibody (CD4): 1 part of CD4 primary antibody (manufacturer: abcam, catalog number: ab133616), 1 part of bovine serum albumin, 0.1 part of sodium azide solution, and 97.9 parts of phosphate buffer.

[0057] Preferably, the non-specific antigen blocking solution includes: 50 parts of goat serum, 49.9 parts of glycerol, and 0.1 parts of sodium azide solution.

[0058] Preferably, the direct-use secondary antibody is HRP-goat anti-rabbit IgG polymer (manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., catalog number: KIT-5005).

[0059] Preferably, DAB color developing agent (manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., product number: DAB-2032).

[0060] Preferably, phosphate buffered saline (PBS): 8 g sodium chloride, 0.2 g potassium chloride, 1.44 g disodium hydrogen phosphate, 0.24 g potassium dihydrogen phosphate, and 800 ml deionized pure water.

[0061] Preferably, Tris buffered saline solution (PH 7.6): 6.05 g of tris(hydroxymethyl)aminomethane, 8.76 g of sodium chloride, and 800 ml of deionized pure water.

[0062] Preferably, the phosphate buffer detergent comprises: 99.5 parts of phosphate buffer and 200.5 parts of polysorbate.

[0063] Preferably, a transparent agent (manufacturer: Zhuhai Besuo Biotechnology Co., Ltd., item number: BA7002D).

[0064] All the above reagents were stored in a 4°C refrigerator away from light. When using, take them out and place them in a place away from sunlight at room temperature. They can be used after 10 minutes of rewarming.

[0065] Example 2

[0066] The use of the staining kit of Example 1 of the present invention in the pathological co-localization evaluation staining of an enteritis model is based on the staining method of the staining kit of Example 1 above, and the specific steps are as follows:

[0067] Step 1: Animal dissection and fixation

[0068] Specimen fixation: clean the intestine with 0.9% saline, roll it into a Swiss roll with the villi facing outward, put the rat intestine into a 24-well cell culture plate and the mouse intestine into a 48-well cell culture plate, fix the specimen with 10% neutral formalin for 6 hours, replace with new fixative and soak for 24 hours.

[0069] Step 2: Specimen processing

[0070] 1) Fine trimming and sampling: flatten the fixed specimen and place it in a tissue embedding box;

[0071] 2) Dehydration and wax dipping: Place in a dehydrator and place in gradient ethanol for 6.5 hours, xylene for 40 minutes, and paraffin for 3 hours;

[0072] 3) Paraffin embedding: Take it out from the dehydrator and put it into the embedding machine for embedding.

[0073] Step 3: Paraffin sectioning

[0074] 1) Paraffin embedding: embed the surface to be sectioned facing downwards, and remove the block after freezing;

[0075] 2) Slice and scoop out slices: Cut a slice with a thickness of 5 microns, spread it in a 45℃ water bath, scoop out the slices and dry them at 60℃ for 30 minutes.

[0076] Step 4: Staining and mounting

[0077] 1) Dewaxing and antigen repair: preheat the vacuum drying oven to 93°C in advance, pour 250 ml of dewaxing antigen repair solution (PH 9.0) into the container and soak the slices for 5 minutes; release the pressure, take out the slices and soak them in 70°C phosphate buffer for 3 times / 5 minutes each time, and wash them in room temperature Tris buffer solution for 3 times / 5 minutes each time;

[0078] 2) Peroxidase blocking: use peroxidase inhibitor to drip or soak at room temperature for 30 minutes, remove the peroxidase inhibitor and soak in phosphate buffer detergent 3 times / 5 minutes each time;

[0079] 3) Primary antibody labeling: incubate with direct-use primary antibody (CD4) at room temperature for 1 hour, and soak in phosphate buffer detergent 3 times / 5 minutes each time;

[0080] 4) Non-specific antigen blocking solution, incubate at room temperature for 30 minutes, and soak in phosphate buffer detergent 3 times / 5 minutes each time;

[0081] 5) Secondary antibody incubation: direct-use secondary antibody HRP-goat anti-rabbit IgG polymer was incubated at room temperature for 15 minutes, and then soaked in phosphate buffer detergent 3 times / 5 minutes each time;

[0082] 6) Color development, DAB color developer (manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., catalog number: DAB-2032) was prepared by taking 1 ml of reagent 1, 50 μl of reagent 2, and 50 μl of reagent 3, and the mixture was allowed to stand for 10 minutes, and then added dropwise to the tissue of the tissue section, and incubated with DAB color developer at room temperature for 5 minutes, and then soaked in phosphate buffer detergent once / 5 minutes each time;

[0083] 7) Alcian blue staining: use Alcian blue staining solution to stain for 5 minutes at room temperature, and wash with pure water to prevent excess blue from coming out;

[0084] 8) Glycogen oxidation: soak or drip periodic acid solution onto the slices and incubate at 2-8°C for 5 minutes, then rinse with deionized / pure water 3 times;

[0085] 9) PAS staining: immerse the sections in Schiff reagent at 2-8°C for 5 minutes, and then rinse with running water for 5-10 minutes until the liquid is colorless;

[0086] 10) For nuclear staining, soak the sections in hematoxylin staining solution for 30 seconds, rinse with running water for 2 minutes, and rinse with pure water 3 times;

[0087] 11) Dry and seal the slides. Air dry naturally or dry at 60°C. Add a transparent agent to make them transparent and then seal them with sealing glue.

[0088] Step 5: Scanning and Slicing Quality Control

[0089] After sealing and drying, the sections were scanned under white light, and the staining results were as follows: Figure 1 Shown: Immunohistochemical staining of immune cell markers (tan), special staining of intestinal secretory cells (blue and purple), all cell nuclei in the tissue (light blue or bluish white), cytoplasm and extracellular matrix staining (red or pink), red blood cells (yellow).

[0090] 3. Experiment

[0091] 3.1 Comparative Experiments with Existing Technologies

[0092] 3.1.1. Comparative Example 1: Comparison between HE staining in the prior art and staining in the present invention

[0093] The wax block slices of step 3 of Example 2 were used to perform the HE staining step of the prior art, and finally the HE staining results of the two slices were compared ( Figure 2 A is the section staining result of HE staining in the prior art; Figure 2 B is the section staining result obtained according to the staining experiment operation of Example 2 of the present invention).

[0094] The specific steps of HE staining in the prior art are as follows:

[0095] Step 1: Staining and mounting

[0096] 1) Paraffin section: Use the wax block of step 3 of Example 2 to slice, 3 μm thick, and bake at 60° C. for 30 minutes after spreading;

[0097] 2) Dewaxing and hydration: Soak in xylene 4 times / 8 minutes each time, anhydrous ethanol 3 times / 5 minutes each time, 95% ethanol 2 times / 5 minutes each time, 75% ethanol 1 time for 1 minute, and rinse with running water for 5 minutes;

[0098] 3) Hematoxylin staining: Soak in hematoxylin staining solution (manufacturer: Harbin Green Specimen Technology Development Co., Ltd., catalog number: GS-02) for 5 minutes and rinse with running water for 10 minutes;

[0099] 4) Hematoxylin differentiation: Soak in hydrochloric acid ethanol for 5 seconds and rinse with running water for 10 minutes;

[0100] 5) Eosin staining: Soak in eosin staining solution (manufacturer: Harbin Green Specimen Technology Development Co., Ltd., catalog number: GS-05) for 20 minutes and rinse with running water for 10 seconds;

[0101] 6) Eosin differentiation: 75% ethanol soaked twice / each time for 10 seconds, 95% ethanol soaked three times / each time for 30 seconds, anhydrous ethanol soaked four times / each time for 1 minute;

[0102] 7) Transparent sealing: Soak in xylene 3 times / 2 minutes each time, and seal with neutral gum.

[0103] Step 2: Scanning and Slicing Quality Control

[0104] After sealing and drying, the sections were scanned under white light, and the staining results were as follows: Figure 2 Shown: Cell nuclei are stained blue or blue-purple by hematoxylin, and cytoplasm and extracellular matrix are stained pink or red by eosin.

[0105] 3.1.2. Comparative Case 2: Comparison between the immunohistochemical staining of the prior art and the staining of the present invention

[0106] The wax block sections of step 3 of Example 2 and the wax block sections after dewaxing and antigen repair in the prior art and the present invention were used to perform the immunohistochemical staining steps of the prior art, and finally the immunohistochemical staining results of the four sections were compared ( Figure 3 A is the section staining result obtained by using the wax block section of step 3 of Example 2 to implement the immunohistochemical staining step of the prior art; Figure 3 B is the section staining result obtained by the staining experiment operation according to Example 2 of the present invention; Figure 3 C is the section staining result of the immunohistochemical staining step of the prior art using the wax block section after dewaxing and antigen repair by the prior art; Figure 3 D is the section staining result of the immunohistochemical staining step of the prior art using the wax block sections after dewaxing and antigen repair of the present invention).

[0107] The method for obtaining the dewaxing and antigen repairing sections in the prior art is specifically as follows:

[0108] 1) Paraffin section: Use the wax block of step 3 of Example 2 to slice, 5 μm thick, and bake at 60° C. for 30 minutes after spreading;

[0109] 2) Dewaxing and hydration: Soak in xylene 4 times / 8 minutes each time, anhydrous ethanol 3 times / 5 minutes each time, 95% ethanol 2 times / 5 minutes each time, 75% ethanol 1 time for 1 minute, and rinse with running water for 5 minutes;

[0110] 3) Antigen retrieval: Treat with antigen retrieval solution (manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., product number: MVS-0050), microwave or pressure cook on high for 20 minutes, then let stand for 30 minutes to expose the target antigen. After the slide is taken out and cooled, it is immersed in phosphate buffered saline (PBS solution) and washed 3 times / 5 minutes each time.

[0111] The method for obtaining the dewaxed and antigen-recovered sections of the present invention is specifically as follows:

[0112] 1) Paraffin section: Use the wax block of step 3 of Example 2 to slice, 5 μm thick, and bake at 60° C. for 30 minutes after spreading;

[0113] 2) Dewaxing and antigen repair: preheat the vacuum drying oven to 93°C in advance, pour 250 ml of dewaxing antigen repair solution (PH9.0) into a container and soak the sections for 5 minutes; release the pressure, take out the sections and soak and wash them in 70°C phosphate buffer for 3 times / 5 minutes each time, and wash them in room temperature Tris buffer solution for 3 times / 5 minutes each time.

[0114] The immunohistochemical staining steps of the prior art are specifically as follows:

[0115] Step 1: Staining and mounting

[0116] 1) Block endogenous enzymes: Incubate with endogenous enzyme blocking agent (manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., catalog number: BLK-0001) for 30 min, and then soak and wash in PBS solution for 3 times / 5 minutes each time;

[0117] 2) Serum blocking: incubate with 5% BSA solution for 1 hour, then remove the 5% BSA solution;

[0118] 3) Primary antibody incubation: freshly prepare PBS and CD4 primary antibody (manufacturer: abcam, catalog number: ab133616) stock solution at a ratio of 50:1 μl, add drops to the slide tissue, incubate at room temperature for 2 hours, and soak and wash in PBS solution 3 times / 5 minutes each time;

[0119] 4) Secondary antibody incubation: freshly prepare PBS, glycerol, and secondary antigen solution (manufacturer: Jackson, catalog number: 115-035-002), mix them at a ratio of 500:500:1 μl, add them dropwise to the slide tissue, incubate at room temperature for 30 minutes, and soak and wash in PBS solution 3 times / 5 minutes each time;

[0120] 5) Color development: DAB color developer (manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., catalog number: DAB-2032) was prepared by taking 1 ml of reagent 1, 50 μl of reagent 2, and 50 μl of reagent 3, and the mixture was allowed to stand for 10 minutes, then added dropwise to the tissue of the tissue section, and incubated with DAB color developer at room temperature for 5 minutes, and then rinsed with running water for 5 minutes;

[0121] 6) Hematoxylin staining and sealing: soak in hematoxylin staining solution for 5 minutes, soak in 75% ethanol twice / 1 minute each time, soak in 95% ethanol three times / 5 minutes each time, soak in anhydrous ethanol four times / 5 minutes each time, soak in xylene three times / 2 minutes each time, and seal with neutral gum.

[0122] Step 2: Scanning and Slicing Quality Control

[0123] After sealing and drying, the sections were scanned under white light, and the staining results were as follows: Figure 3 As shown: The DAB staining results of positive cells or proteins appear brown, and the non-positive cell nuclei are blue.

[0124] 3.1.3. Comparative Example 3: Comparison between the co-staining of Alcian blue and PAS in the prior art and the staining of the present invention

[0125] The wax block sections of step 3 of Example 2 were used to perform the prior art Alcian blue and PAS co-staining staining steps, and finally the histochemical staining results of the two sections were compared ( Figure 4 A is the section staining result of Alcian blue and PAS co-staining in the prior art; Figure 4 B is the section staining result obtained according to the staining experiment operation of Example 2 of the present invention).

[0126] The prior art Alcian blue and PAS co-staining steps are as follows:

[0127] Step 1: Staining and mounting

[0128] 1) Paraffin section: Use the wax block of step 3 of Example 2 to slice, 5 μm thick, and bake at 60° C. for 30 minutes after spreading;

[0129] 2) Dewaxing and hydration: Soak in xylene 4 times / 8 minutes each time, anhydrous ethanol 3 times / 5 minutes each time, 95% ethanol 2 times / 5 minutes each time, 75% ethanol 1 time for 1 minute, and rinse with running water for 5 minutes;

[0130] 3) Alcian blue staining: Use Alcian blue staining solution to stain for 60 minutes at room temperature, and then wash with pure water until no excess blue is removed;

[0131] 4) Glycogen oxidation: Soak or drip periodic acid solution onto the slices and incubate at room temperature for 20 minutes, then rinse with deionized water three times;

[0132] 5) PAS staining: Soak the sections in Schiff reagent and incubate at room temperature for 60 minutes, then rinse with running water for 15 minutes until the liquid is colorless;

[0133] 6) Drying and sealing: Soak in xylene 3 times / 2 minutes each time, and seal with neutral gum.

[0134] Step 2: Scanning and Slicing Quality Control

[0135] After sealing and drying, the sections were scanned under white light, and the staining results were shown in Figure 4: Alcian blue can stain the acidic mucopolysaccharide in the cytoplasm into blue, and PAS staining can stain the neutral mucin into deep red or red-purple.

[0136] 3.1.4. Comparative Case 4: Comparison between the dewaxing and antigen retrieval in the prior art and the dewaxing and antigen retrieval in the present invention

[0137] Step 1: Deparaffinization and antigen retrieval using existing techniques

[0138] 1) Paraffin section: Use the wax block of step 3 of Example 2 to slice, 5 μm thick, and bake at 60° C. for 30 minutes after spreading;

[0139] 2) Dewaxing and hydration: Soak in xylene 4 times / 8 minutes each time, anhydrous ethanol 3 times / 5 minutes each time, 95% ethanol 2 times / 5 minutes each time, 75% ethanol 1 time for 1 minute, and rinse with running water for 5 minutes;

[0140] 3) Antigen retrieval: Treat with antigen retrieval solution (manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., catalog number: MVS-0050), microwave or pressure cook on high for 20 minutes, then let stand for 30 minutes to expose the target antigen. After the slide is taken out and cooled, soak in PBS solution and wash 3 times / 5 minutes each time.

[0141] Step 2: Dewaxing and antigen retrieval of the present invention

[0142] 1) Paraffin section: Use the wax block of step 3 of Example 2 to slice, 5 μm thick, and bake at 60° C. for 30 minutes after spreading;

[0143] 2) Dewaxing and antigen repair: preheat the vacuum drying oven to 93°C in advance, pour 250 ml of dewaxing antigen repair solution PH9.0 (manufacturer: Fuzhou Maixin Biotechnology Development Co., Ltd., item number: DAS-0021) into a container and soak the slices for 5 minutes; release the pressure, take out the slices and soak them in 70°C phosphate buffer for 3 times / 5 minutes each time, and wash them in room temperature Tris buffer solution for 3 times / 5 minutes each time.

[0144] Step 3: Compare the dewaxing conditions of the existing method and the present invention, observe the clarity and transparency of the two slices with the naked eye to see whether the tissue is completely exposed to the air; observe the two slices under a microscope to see whether there are wax spots or unclear tissues; the results are as follows: Figure 5 As shown ( Figure 5 A and 5B are the sections before dewaxing in the prior art and before dewaxing in the present invention, respectively; Figure 5 C and 5D are the sections after dewaxing in the prior art and the present invention, respectively).

[0145] Experimental conclusion: Comparison between Example 2 and Comparative Cases 1, 2, and 3 shows that no difference in tissue morphological structure positioning and identification was found in HE staining, immunohistochemical staining, and Alcian blue and PAS co-staining, and the corresponding staining results can be observed.

[0146] In Example 2, corresponding pathological diagnostic indicators can be observed, including diarrhea, mucoid stools, mucosal ulcers, intestinal wall thickening, fibrotic granulomas, angiogenesis, edema, inflammatory infiltration, goblet cell secretory cell reduction or abnormal secretion affecting digestive flora disorders, etc., and compared with the pathological diagnosis results of comparative cases 1, 2, and 3, the positioning is better, and the result difference and positioning position determination are more appropriate.

[0147] It can be seen from Comparative Case 2 and Comparative Case 4 that there is no significant difference in the dewaxing between the prior art and the present invention; however, it can be seen from the antigen repair method of the present invention that the histochemical positive results are more, the signal is stronger, and the tissue section integrity is better than the antigen repair method of the prior art.

[0148] Staining comparison results: When Example 2 was compared with Comparative Examples 1, 2, and 3, no difference was found in HE staining, immunohistochemical staining, and co-staining of Alcian blue and PAS.

[0149] Advantages of pathological diagnosis: The pathological diagnosis indicators of Example 2 are clear, and the positioning is better than that of comparative cases 1, 2, and 3.

[0150] Antigen repair effect: From Comparative Case 2 and Comparative Case 4, it can be seen that the antigen repair method of the present invention has more positive results, stronger signals, and intact tissues.

[0151] 3.2 Stability / Repeatability Verification of the Kit of the Present Invention

[0152] 3.2.1. Using the same kit as in Example 1 and the same method as in Example 2, two different groups of people conducted the experiment. The dried slides obtained by the two different groups of people were scanned with white light. The staining results were as follows: Figure 6 Shown: Immunohistochemical staining of immune cell markers (tan), special staining of intestinal secretory cells (blue and purple), all cell nuclei in the tissue (light blue or bluish white), cytoplasm and extracellular matrix staining (red or pink), red blood cells (yellow).

[0153] 3.2.2. The paraffin sections and staining reagents of step 3 of Example 2 after storage for 6 months were used, and the same operation method as in Example 2 was used to perform white light scanning on the obtained dried mounts, and the deviation of the two staining results was compared to observe the performance stability of the kit. The staining results were as follows: Figure 7 Shown: Immunohistochemical staining of immune cell markers (tan), special staining of intestinal secretory cells (blue and purple), all cell nuclei in the tissue (light blue or bluish white), cytoplasm and extracellular matrix staining (red or pink), red blood cells (yellow).

[0154] Experimental results: In step 3.2.1 and step 3.2.2, the staining kit of the present invention was verified again by different personnel and different storage times. The results of the two experiments were consistent in the personnel comparison, and no obvious staining difference was found. The staining results in the retained sample retest were consistent with the staining results 6 months ago, and no staining difference was found.

[0155] The experiments in step 3.2.1 and step 3.2.2 above verified that the staining kit of the present invention was consistent in the comparison results between personnel and time, and the staining method had excellent repeatability, and could be extended to more institutions to use the staining kit and application of the present invention.

[0156] The above-mentioned embodiments only express several implementation methods of the present invention, and the description thereof is relatively specific and detailed, but it cannot be understood as limiting the scope of the present invention. It should be pointed out that, for ordinary technicians in this field, several variations and improvements can be made without departing from the concept of the present invention, which all belong to the protection scope of the present invention.

Claims

1. A staining kit for pathological co-localization evaluation of enteritis model, characterized in that: The staining kit includes the following components: hematoxylin staining solution, Alcian blue staining solution, periodic acid solution, Schiff reagent, dewaxing antigen repair solution, peroxidase inhibitor, direct-use primary antibody CD4, non-specific antigen blocking solution, direct-use secondary antibody HRP-goat anti-rabbit IgG polymer, DAB color developer, phosphate buffer, Tris buffered saline solution, phosphate buffer detergent, and transparent agent.

2. A staining kit for pathological co-localization evaluation of enteritis model according to claim 1, characterized in that: The Alcian blue staining solution is a 1% Alcian blue staining solution, and the 1% Alcian blue staining solution is obtained by adding 1g of Alcian blue 8GX powder to 3ml of acetic acid and 97ml of distilled water, fully dissolving the solution, and standing for 1 hour to obtain the solution.

3. A staining kit for pathological co-localization evaluation of enteritis model according to claim 1, characterized in that: The periodic acid solution is obtained by adding 0.5 g of periodic acid to 100 ml of water and allowing the solution to stand for 30 minutes.

4. A staining kit for pathological co-localization evaluation of enteritis model according to claim 1, characterized in that: The Schiff reagent is prepared from basic fuchsin 1g, 1mol / L hydrochloric acid 20ml, sodium metabisulfite 1.5g, distilled water 200ml, and activated carbon 2g; The Schiff reagent is obtained by dissolving 1 g of basic fuchsin in 200 ml of distilled water at 80° C., heating and boiling for a while, and fully stirring, filtering when cooled to 50° C., adding 20 ml of 1 mol / L hydrochloric acid, and adding 1.5 g of sodium metabisulfite when cooled to 25° C., sealing and placing in a dark place at room temperature for 24 hours, at which time the solution is light khaki or light red, then adding 2 g of activated carbon powder, fully shaking the solution, and filtering after standing for 2 hours to obtain a colorless, clear and transparent solution.

5. A staining kit for pathological co-localization evaluation of enteritis model according to claim 1, characterized in that: The peroxidase inhibitor is prepared by preparing 10 parts of 30% by mass hydrogen peroxide and 90 parts of phosphate buffer.

6. A staining kit for pathological co-localization evaluation of enteritis model according to claim 1, characterized in that: The direct-use primary antibody CD4 is prepared from 1 part of CD4 primary antibody, 1 part of bovine serum albumin, 0.1 part of sodium azide solution, and 97.9 parts of phosphate buffer.

7. A staining kit for pathological co-localization evaluation of enteritis model according to claim 1, characterized in that: The nonspecific antigen blocking solution is prepared from 50 parts of goat serum, 49.9 parts of glycerol and 0.1 parts of sodium azide solution.

8. A staining kit for pathological co-localization evaluation of enteritis model according to claim 1, characterized in that: The phosphate buffer is prepared from 8 g of sodium chloride, 0.2 g of potassium chloride, 1.44 g of disodium hydrogen phosphate, 0.24 g of potassium dihydrogen phosphate, and 800 ml of deionized pure water.

9. A staining kit for pathological co-localization evaluation of enteritis model according to claim 1, characterized in that: The Tris buffered saline solution is prepared from 6.05 g of tris(hydroxymethyl)aminomethane, 8.76 g of sodium chloride, and 800 ml of deionized pure water; The phosphate buffer detergent is prepared from 99.5 parts of phosphate buffer and 200.5 parts of polysorbate.

10. A dyeing method based on the dyeing kit according to any one of claims 1 to 9, characterized in that: The specific steps are: 1) Preparation of paraffin sections of animal tissues; 2) Dewaxing and antigen repair: preheat the vacuum drying oven to 93°C in advance, pour in 250 ml of dewaxing antigen repair solution and soak the slices obtained in step 1) for 5 minutes; take out the slices and soak them in 70°C phosphate buffer for 3 times / 5 minutes each time, and wash them in room temperature Tris buffer solution for 3 times / 5 minutes each time; 3) Peroxidase blocking: the slices treated in step 2) are blocked by dripping or soaking with a peroxidase inhibitor at room temperature for 30 minutes, the peroxidase inhibitor is removed and the slices are soaked in a phosphate buffer detergent 3 times / 5 minutes each time; 4) Primary antibody labeling: the slices treated in step 3) were incubated with direct-use primary antibody CD4 at room temperature for 1 hour, and then soaked in phosphate buffer detergent 3 times / 5 minutes each time; 5) The slices treated in step 4) were incubated in non-specific antigen blocking solution at room temperature for 30 minutes, and then soaked in phosphate buffer detergent 3 times / 5 minutes each time; 6) Secondary antibody incubation: the slices treated in step 5) were incubated with direct secondary antibody HRP-goat anti-rabbit IgG polymer at room temperature for 15 minutes, and then soaked in phosphate buffer detergent 3 times / 5 minutes each time; 7) DAB color development: the slices treated in step 6) were incubated with DAB color developer for 5 min at room temperature, and then soaked in phosphate buffer detergent once for 5 min each time; 8) Alcian blue staining: the slices treated in step 7) are stained with Alcian blue staining solution at room temperature for 5 minutes, and then washed with pure water until no excess blue is removed; 9) Glycogen oxidation: the slices treated in step 8) are immersed in periodic acid solution or dripped onto the slices and incubated at 2-8°C for 5 minutes, and then washed with deionized / pure water for 3 times; 10) PAS staining: the slices treated in step 9) are immersed in Schiff reagent and incubated at 2-8°C for 5 minutes, and then rinsed with running water for 5-10 minutes until the liquid is colorless; 11) Nuclear staining: the sections treated in step 10) were immersed in hematoxylin staining solution for 30 seconds, rinsed with running water for 2 minutes, and then rinsed with pure water for 3 times; 12) Drying and sealing. The slices treated in step 11) are air-dried or dried at 60° C., and transparent agent is added to make them transparent, and then the slices are sealed with sealing glue.

Images