Prostate lesion tissue immunofluorescence detection kit and detection method

Abstract

The present invention proposes a prostate lesion tissue immunofluorescence detection kit and detection method, which belong to the technical field of kits. It is prepared from the following raw materials by weight: 40-60 parts of TYR-xxxPlus fluorescent dye, 30-45 parts of TSA Buffer, 20-40 parts of Polymer-HRP monoclonal antibody mouse / rabbit secondary antibody, 15-30 parts of antibody, 5-15 parts of Dapi dye solution, 50-70 parts of antibody diluent, and 50-70 parts of peroxidase blocking solution. This kit adopts a non-biotin amplification system, which greatly shortens the experimental time compared with traditional double staining experiments and effectively avoids the interference of endogenous biotin. It is suitable for the diagnosis and differential diagnosis of prostate cancer, PIN, and AAH in prostate resection specimens, needle biopsy specimens, electroresection specimens, and transurethral resection specimens, and is particularly suitable for conventional formalin-fixed paraffin-embedded sections.

Description

Technical Field

[0001] The present invention relates to the technical field of kits, and in particular to a prostate lesion tissue immunofluorescence detection kit and a detection method. Background Art

[0002] Prostate disease is a common disease in elderly men. Clinically, prostate lesions mainly include prostatitis, benign prostatic hyperplasia and prostate cancer. Its diagnosis depends largely on the pathological examination of puncture biopsy specimens, and immunohistochemistry plays an important role in its pathological auxiliary diagnosis. Conventional immunohistochemistry can only stain one antibody on a slice. However, due to the small amount of tissue in the puncture biopsy specimen, when there are too few tumors / suspicious cells in the specimen, conventional immunohistochemistry often causes certain difficulties in pathological diagnosis.

[0003] P504s is a specific marker for prostate cancer. It is highly expressed in prostate cancer and not expressed in normal prostate tissue. However, it may be weakly expressed in prostate intraepithelial neoplasia (PIN) and atypical hyperplasia (AAH) tissue. p63 and HMWCK mark the nucleus and cytoplasm of prostate basal cells respectively. The combination of the two can improve the recognition of basal cells. Normal prostate glands have two layers of cells (basal cells and glandular epithelial cells), while prostate cancer has no or only a small amount of basal cells. In this way, the use of three combined antibodies can directly observe various lesions in the same section, which is convenient for comparison and is conducive to the diagnosis and differential diagnosis of prostate cancer, PIN and AHH. It is suitable for the examination of excisional biopsy and puncture biopsy specimens, especially for difficult cases.

[0004] The existing prostate lesion tissue immunohistochemistry double staining kit uses a combination of monoclonal antibodies and dual enzyme labeling, which can detect three antigens in a prostate tissue section at the same time. However, since this technology uses ready-to-use combination antibodies, it requires that the antibody sources must be from different species, and rabbit monoclonal antibodies with strong specificity and affinity cannot be used. Moreover, the corresponding antibodies cannot be used for other studies and will cause cyclic staining. The experimental cycle is longer than that of the prostate lesion tissue immunohistochemistry double staining kit. Summary of the invention

[0005] The purpose of the present invention is to provide a prostate lesion tissue immunofluorescence detection kit and detection method, which adopts a non-biotin amplification system, greatly shortens the experimental time compared with the traditional double staining experiment, and effectively avoids the interference of endogenous biotin. It is suitable for the diagnosis and differential diagnosis of prostate cancer, PIN, AAH in prostate resection specimens, needle biopsy specimens, electroresection specimens, and transurethral resection specimens, especially for conventional formalin-fixed paraffin-embedded sections.

[0006] The technical solution of the present invention is achieved in this way:

[0007] The invention provides a prostate lesion tissue immunofluorescence detection kit, which is prepared from the following raw materials by weight: 40-60 parts of TYR-xxxPlus fluorescent dye, 30-45 parts of TSA Buffer, 20-40 parts of Polymer-HRP monoclonal antibody mouse / rabbit secondary antibody, 15-30 parts of antibody, 5-15 parts of Dapi dye solution, 50-70 parts of antibody diluent, and 50-70 parts of peroxidase blocking solution.

[0008] As a further improvement of the present invention, the TYR-xxxPlus fluorescent dye is selected from any three of TYR-480Plus fluorescent dye, TYR-520Plus fluorescent dye, TYR-570Plus fluorescent dye, TYR-620Plus fluorescent dye, TYR-690Plus fluorescent dye, and TYR-780Plus fluorescent dye; the antibodies include primary antibody P504S, primary antibody P63, and primary antibody CK, with a mass ratio of 10:12-15:10.

[0009] As a further improvement of the present invention, the antibody diluent is prepared from the following raw materials in parts by weight: 80-100 parts of PBS buffer, 0.5-1 part of bovine serum albumin, 0.5-2 parts of trehalose, 20-40 parts of glycerol, 0.5-1 part of surfactant, and 0.1-0.5 part of preservative.

[0010] As a further improvement of the present invention, the surfactant is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85.

[0011] As a further improvement of the present invention, the preparation method of the preservative is as follows:

[0012] S1. Preparation of lauric acid vitamin C ester: lauric acid and vitamin C were added to toluene, concentrated sulfuric acid was added, and the reaction mixture was heated to reflux, and the reaction solution was washed with saturated sodium carbonate and water in turn, and the organic layer was collected and separated by column chromatography to obtain lauric acid vitamin C ester;

[0013] S2. Preparation of carboxylated chitosan: dissolving chitosan in an acid solution, adding α-ketoglutaric acid, heating and stirring the reaction, adding sodium borohydride, stirring the reaction, adding ethanol for precipitation, washing, and drying to obtain carboxylated chitosan;

[0014] S3. Preparation of preservative: Carboxylated chitosan and lauric acid vitamin C ester are added to toluene, concentrated sulfuric acid is added, the mixture is heated under reflux for reaction, ethanol is added for precipitation, the mixture is washed, and the mixture is dried to obtain a preservative.

[0015] As a further improvement of the present invention, the molar ratio of lauric acid to vitamin C in step S1 is 1:0.9-1, the amount of concentrated sulfuric acid added is 1-2wt% of the total mass of the system, and the heating reflux reaction time is 3-5h; the acid solution in step S2 is 2-3wt% acetic acid or lactic acid, the mass ratio of chitosan, α-ketoglutaric acid, and sodium borohydride is 10:14-17:3-5, the temperature of the heating and stirring reaction is 35-40°C, the time is 20-24h, and the stirring reaction time is 10-12h; the mass ratio of carboxylated chitosan, lauric acid vitamin C ester, and concentrated sulfuric acid in step S3 is 10:3-4:0.5-1, and the heating reflux reaction time is 4-6h.

[0016] The present invention further protects a method for detecting prostate lesion tissue using the above-mentioned prostate lesion tissue immunofluorescence detection kit, comprising the following steps:

[0017] (1) Preparation of cell tissue sections;

[0018] (2) Perform antigen retrieval on cell tissue sections;

[0019] (3) Place the sections in a 2-4wt% hydrogen peroxide solution and incubate at room temperature in the dark for 10-20 min. Place the slides in a PBS solution and wash by shaking on a decolorizing shaker.

[0020] (4) After the slices are dried, use a tissue pen to draw a circle around the tissue, add normal goat serum for blocking inside the circle to evenly cover the tissue, and block at room temperature for 25-35 minutes;

[0021] (5) Discard the blocking solution, add the primary antibody X diluted with antibody diluent onto the slices, and place the slices flat in a light-proof humidified box for incubation;

[0022] (6) Incubation with poly-HRP secondary antibody: Place the slide in PBS solution and shake on a decolorizing shaker to wash. After the slide is dried, add poly-HRP secondary antibody of the same species as the primary antibody to cover the tissue. Incubate at room temperature away from light and wash with PBS solution.

[0023] (7) Mix the fluorescent dye and TSA buffer evenly according to the mass ratio of the kit to prepare the TSA fluorescent dye reaction solution, add it dropwise to the slices, evenly cover the tissue, react at room temperature for 5-10 minutes, and wash with PBS solution;

[0024] (8) Place the paraffin sections in antigen retrieval solution or antibody elution solution to cover the samples, react at 36-38℃ for 5-20min, discard the elution solution, add an appropriate amount of antibody elution solution to cover the samples again, react at 36-38℃ for 5-20min, discard the elution solution, and wash with PBS solution;

[0025] (9) Repeat steps (2) to (8) using another fluorescent dye to perform a second round of labeling;

[0026] (10) Repeat steps (2) to (8) using another fluorescent dye to perform a third round of labeling;

[0027] (11) Place the slide in PBS solution and shake on a decolorizing shaker to wash. After the slices are dried, add Dapi staining solution to the circle and incubate at room temperature in the dark for 5 min-20 min.

[0028] (12) Place the slide in PBS solution and shake on a decolorizing shaker to wash. After the slices are dried, seal them with an anti-fluorescence quenching sealant.

[0029] (13) Observe the slices under an imaging system, collect images, and analyze the results.

[0030] As a further improvement of the present invention, the antigen repair method in step (2) includes: 10-15 MPa pressure for 1-2 minutes, then boiling at 37°C for 15 minutes, or 90-100°C water bath for 15-25 minutes, or microwave oven at medium heat for 5-10 minutes, stop the heat for 5-10 minutes, and switch to medium-low heat for 4-8 minutes.

[0031] As a further improvement of the present invention, the incubation in step (5) is incubation at 4°C overnight or heating treatment at 37°C for 1-2 hours.

[0032] As a further improvement of the present invention, the primary antibody X is primary antibody P504S, primary antibody P63 or primary antibody CK, and the poly-HRP secondary antibody is poly-HRP goat anti-mouse / rabbit universal secondary antibody, which is ready-to-use AFIHC001.

[0033] Technical principle of the kit: The present invention uses HRP-labeled secondary antibodies. In the presence of hydrogen peroxide, HRP catalyzes the tyramide fluorescein substrate added to the system to generate an activated fluorescent substrate. The activated fluorescein substrate can covalently bind to the tyrosine residues on the antigen to form a stable antigen-tyramide fluorescent substrate complex. After that, the non-covalently bound antibodies are washed away with heat repair, and then a primary antibody is replaced for a second round of incubation, and another fluorescein substrate is replaced. This reciprocating process can achieve triple labeling. One of the advantages of this kit is that the fluorescent signal is more than 10 times stronger than the traditional immunohistochemistry signal, and multiple labeling can be achieved using the same primary antibody.

[0034] The detailed principle of TSA is to utilize the peroxidase reaction of tyramide. Tyramide salt forms a covalent binding site under the catalysis of HRP (in the presence of H202), producing a large amount of enzymatic products. This product can bind to the surrounding protein residues (including tryptophan, histidine and tyrosine residues), so that a large amount of fluorescein is deposited at the antigen-antibody binding site, resulting in a 10-100-fold enhancement of the detection signal.

[0035] This method of multiple immunofluorescence uses the HRP on the secondary antibody (rather than directly coupling fluorescein) to catalyze the inactive fluorescein added to the system later. Fluorescent is activated by HRP and hydrogen peroxide, and covalently couples to the tyrosine residues of the adjacent protein, so that the protein sample and fluorescein are stably bound. Then, the non-covalently bound antibodies in the previous round are washed away by heat repair treatment such as microwave, boiling or water bath, and the covalently bound fluorescein is stably covalently bound to the sample slice protein. Then, a new primary antibody is used for the second round of incubation, and the cycle repeats. After all the antibody incubations are completed and the fluorescein is well bound, the results are finally detected. Since only a single antibody is incubated in the system each time, there is no need to worry about antibody cross-reactions and the species matching of the primary and secondary antibodies, which greatly reduces the restrictions on the selection and matching of antibodies of different species during experimental design. In other words, if TSA technology is used, all targets on the same slide can use highly specific rabbit monoclonal antibodies. Experiments can be carried out with the same anti-rabbit poly-HRP secondary antibody, and the signal amplification multiple is greatly enhanced.

[0036] The present invention has the following beneficial effects:

[0037] The invention prepares a preservative which is prepared from natural preservatives, has high safety, has good protection for antibodies and prevents bacterial damage, can be stored for a long time, and improves the activity of antibodies.

[0038] The lauric acid and vitamin C are subjected to an esterification reaction to prepare lauric acid vitamin C ester, which not only greatly improves the antibacterial, antioxidant and anti-inflammatory effects of the components, improves the resistance to bacteria and pathogens, but also reduces the influence of harmful substances on antibodies. However, the antibacterial property is insufficient, the activity of the antibodies cannot be maintained for a long time, and the lauric acid vitamin C ester has no protection and wetting properties for proteins.

[0039] The invention adopts chitosan which is carboxylated and then coupled with lauric acid vitamin C ester, which greatly improves the antibacterial property of the prepared preservative, and at the same time, has a good protective effect on proteins and good wettability, thereby greatly improving the efficacy of the antibody diluent, maintaining the stability of multiple antibody molecules, maintaining the biological activity of the antibody to the greatest extent, extending the shelf life of the antibody molecules, and reducing the non-specific binding of the antibody during use.

[0040] This kit uses a non-biotin amplification system, which greatly shortens the experimental time compared to traditional double-staining experiments and effectively avoids the interference of endogenous biotin. It is suitable for the diagnosis and differential diagnosis of prostate cancer, PIN, and AAH in prostate resection specimens, needle biopsy specimens, electroresection specimens, and transurethral resection specimens, especially for conventional formalin-fixed paraffin-embedded sections. BRIEF DESCRIPTION OF THE DRAWINGS

[0041] In order to more clearly illustrate the embodiments of the present invention or the technical solutions in the prior art, the drawings required for use in the embodiments or the description of the prior art will be briefly introduced below. Obviously, the drawings described below are only some embodiments of the present invention. For ordinary technicians in this field, other drawings can be obtained based on these drawings without paying creative labor.

[0042] Figure 1 The result diagram of the test sample obtained by the method in Example 1 is shown;

[0043] Figure 2 The result diagram of the test sample obtained by the method in Example 2 is shown;

[0044] Figure 3 The result diagram of the test sample obtained by the method in Example 3 is shown;

[0045] Figure 4 This is a result diagram of the test sample obtained by the method in Comparative Example 1;

[0046] Figure 5 This is a graph showing the results of testing samples using the kit prepared by the method in Comparative Example 2. DETAILED DESCRIPTION

[0047] The technical solutions in the embodiments of the present invention are described clearly and completely below. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of them. 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.

[0048] Primary antibody P504S (cat. no.: AF20158), mouse, 1:2000, primary antibody P63 (cat. no.: AF20081), mouse, 1:2000, primary antibody CK (cat. no.: AF20319), rabbit, 1:1000; ready-to-use (cat. no.: AFIHC001) poly-HRP goat anti-mouse / rabbit universal secondary antibody, HMW CK (cat. no.: AF20319), all purchased from Hunan Aifang Biological Co., Ltd.; PBS buffer, pH=7.4, triton-X100 permeabilization buffer, EDTA alkaline antigen retrieval buffer, pH=9.0, TSA buffer, TYR-480Plus fluorescent dye, TYR-520Plus fluorescent dye, TYR-570Plus fluorescent dye, TYR-620Plus fluorescent dye, TYR-690Plus fluorescent dye, TYR-780Plus fluorescent dye, Dapi staining solution, peroxidase blocking solution, and anti-fluorescence quenching sealing agent are all commercially available.

[0049] Preparation Example 1 The preparation method of the preservative is as follows:

[0050] S1. Preparation of lauric acid vitamin C ester: 0.1 mol lauric acid and 0.09 mol vitamin C were added to 500 mL toluene, and concentrated sulfuric acid was added. The amount of concentrated sulfuric acid added was 1 wt% of the total mass of the system. The reaction mixture was heated under reflux for 3 h. The reaction solution was washed with saturated sodium carbonate and water in turn. The organic layer was collected and separated by column chromatography (the volume ratio of ethyl acetate to petroleum ether was 1:1) to obtain lauric acid vitamin C ester; 1H-NMR (300 MHz, DMSO- d 6) δ: 0.5 (-CH3), 1.3 (-CH2-), 1.9-1.2 (CH2-CO), 2.1 (CO-CH2), 2.5 (DMSO), 4.4-4.7 (O-CH2-CH), 4.7 (CH), 11.1 (C3-OH).

[0051] S2. Preparation of carboxylated chitosan: 10 g of chitosan was dissolved in 500 mL of 2 wt% acetic acid solution, 14 g of α-ketoglutaric acid was added, the mixture was heated to 35°C, stirred and reacted for 20 h, 3 g of sodium borohydride was added, stirred and reacted for 10 h, ethanol was added until the ethanol content of the system was 80 wt%, the mixture was precipitated for 1 h, washed, and dried to obtain carboxylated chitosan;

[0052] The infrared spectrum characterization results are as follows: 3442cm -1 Strong -OH absorption bands (OH stretching vibration and NH stretching vibration) appeared near 1610cm -1 (-NH- stretching vibration), 1425cm -1 (CH bending vibration), 1410cm -1(-COO- symmetric stretching vibration), 1090cm -1 (CO absorption peak on the ring); 1642cm -1 (Stretching vibration of C=O in -NH-C=O), 2933cm -1 (CH stretching vibration), indicating that carboxylated chitosan was successfully synthesized.

[0053] S3. Preparation of preservative: 10 g of carboxylated chitosan and 3 g of lauric acid vitamin C ester were added to 500 mL of toluene, 0.5 g of concentrated sulfuric acid was added, and the mixture was heated under reflux for 4 h. Ethanol was added until the ethanol content of the system was 80 wt %, and the mixture was precipitated for 1 h. The mixture was washed and dried to obtain a preservative.

[0054] The infrared spectrum characterization results are as follows: 3410cm -1 -OH absorption bands appeared nearby, but were weaker than those of carboxylated chitosan (OH stretching vibration and NH stretching vibration); 2850cm -1 (Alkyl chain absorption peak), 1614 cm -1 (-NH- stretching vibration), 1425-1430cm -1 (CH bending vibration), 1407cm -1 (-COO- symmetric stretching vibration), 1270 cm -1 (Stretching vibration of -OH on vitamin C), 1088 cm -1 (CO absorption peak on the ring); 1645cm -1 (Stretching vibration of C=O in -NH-C=O), 2910cm -1 (CH stretching vibration), indicating that carboxylated chitosan and lauric acid vitamin C ester were successfully coupled.

[0055] Preparation Example 2 The preparation method of the preservative is as follows:

[0056] S1. Preparation of lauric acid vitamin C ester: 0.1 mol of lauric acid and 0.1 mol of vitamin C were added to 500 mL of toluene, and concentrated sulfuric acid was added. The amount of concentrated sulfuric acid added was 2 wt% of the total mass of the system. The reaction mixture was heated under reflux for 5 h. The reaction solution was washed with saturated sodium carbonate and water in turn. The organic layer was collected and separated by column chromatography (the volume ratio of ethyl acetate: petroleum ether was 1:1) to obtain lauric acid vitamin C ester.

[0057] S2. Preparation of carboxylated chitosan: 10 g of chitosan was dissolved in 500 mL of 3 wt% lactic acid solution, 17 g of α-ketoglutaric acid was added, the mixture was heated to 40°C, stirred for 24 h, 5 g of sodium borohydride was added, stirred for 12 h, ethanol was added until the ethanol content of the system was 80 wt%, the mixture was precipitated for 1 h, washed, and dried to obtain carboxylated chitosan;

[0058] S3. Preparation of preservative: 10 g of carboxylated chitosan and 4 g of lauric acid vitamin C ester were added to 500 mL of toluene, 1 g of concentrated sulfuric acid was added, and the mixture was heated under reflux for 6 h. Ethanol was added until the ethanol content of the system was 80 wt %, and the mixture was precipitated for 1 h. The mixture was washed and dried to obtain a preservative.

[0059] Preparation Example 3 The preparation method of the preservative is as follows:

[0060] S1. Preparation of lauric acid vitamin C ester: 0.1 mol lauric acid and 0.095 mol vitamin C were added to 500 mL toluene, and concentrated sulfuric acid was added. The amount of concentrated sulfuric acid added was 1.5 wt% of the total mass of the system. The reaction mixture was heated under reflux for 4 h. The reaction solution was washed with saturated sodium carbonate and water in turn. The organic layer was collected and separated by column chromatography (the volume ratio of ethyl acetate: petroleum ether was 1:1) to obtain lauric acid vitamin C ester.

[0061] S2. Preparation of carboxylated chitosan: 10 g of chitosan was dissolved in 500 mL of 2.5 wt% acetic acid solution, 15 g of α-ketoglutaric acid was added, the mixture was heated to 37 ° C, stirred for 22 h, 4 g of sodium borohydride was added, stirred for 11 h, ethanol was added until the ethanol content of the system was 80 wt%, precipitated for 1 h, washed, and dried to obtain carboxylated chitosan;

[0062] S3. Preparation of preservative: 10 g of carboxylated chitosan and 3.5 g of lauric acid vitamin C ester were added to 500 mL of toluene, 0.7 g of concentrated sulfuric acid was added, and the mixture was heated under reflux for 5 h. Ethanol was added until the ethanol content of the system was 80 wt %, and the mixture was precipitated for 1 h. The mixture was washed and dried to obtain a preservative.

[0063] Comparative Preparation Example 1

[0064] Compared with Preparation Example 3, the difference is that the preservative is vitamin C laurate.

[0065] Comparative Preparation Example 2

[0066] Compared with Preparation Example 3, the difference is that the preservative is carboxylated chitosan.

[0067] Test Example 1

[0068] 1 mL of the preservative solution (0.1 mg / mL) prepared in Preparation Example 1-3 or Comparative Preparation Example 1-2 was added with 1 mL of DPPH with a concentration of 0.2 mmol / L, and the solution was allowed to stand at room temperature for 30 minutes, and then the absorbance change was measured at a wavelength of 517 nm.

[0069] DPPH clearance rate (%) = [1-(A i -A j ) / A0]×100%

[0070] Where: A i A is the absorbance of 1mL DPPH + 1mL sample solution; j It is the absorbance of 1mL solvent + 1mL test solution; A0 is the absorbance of 1mL DPPH + 1mL solvent (water).

[0071] Vitamin C (0.1 mg / mL) was used as the positive control group.

[0072] The results are shown in Table 1.

[0073] Table 1

[0074]

[0075] It can be seen from the above table that the preservatives prepared in Preparation Examples 1-3 of the present invention have good antioxidant activity.

[0076] Test Example 2

[0077] The test strains were Staphylococcus aureus (ATCC6538), Escherichia coli (ATCC25922) and Candida albicans (ATCC10231). The above strains were selected for the 4th to 6th generations, and the bacterial suspension was prepared with PBS solution with pH=7.4, and the bacterial content was 5×10 5 cfu / mL. The test was conducted in accordance with the 2002 edition of the Technical Specification for Disinfection, using the preservative solution (0.1 mg / mL) prepared in Preparation Example 1-3 and Comparative Preparation Example 1-2, with an action time of 1 min, repeated 3 times, and the test temperature of 25±1°C, and the average value was taken. The results are shown in Table 2.

[0078] Table 2

[0079]

[0080] It can be seen from the above table that the preservatives prepared in Preparation Examples 1-3 of the present invention have good antibacterial effects.

[0081] Example 1. This example provides a prostate lesion tissue immunofluorescence detection kit, which is prepared by the following raw materials in parts by weight: 40 parts of TYR-xxxPlus fluorescent dye, 30 parts of TSA Buffer, 20 parts of Polymer-HRP monoclonal antibody mouse / rabbit secondary antibody, 15 parts of antibody, 5 parts of Dapi dye solution, 50 parts of antibody diluent, and 50 parts of peroxidase blocking solution. The antibodies include primary antibody P504S, primary antibody P63, and primary antibody CK, with a mass ratio of 10:12:10.

[0082] The TYR-xxxPlus fluorescent dye includes TYR-480Plus fluorescent dye, TYR-520Plus fluorescent dye, and TYR-570Plus fluorescent dye, and the mass ratio is 1:1:1.

[0083] The antibody diluent is prepared from the following raw materials by weight: 80 parts of PBS buffer, 0.5 parts of bovine serum albumin, 0.5 parts of trehalose, 20 parts of glycerol, 0.5 parts of Tween-20, and 0.1 parts of the preservative prepared in Preparation Example 1. The preparation method is to mix the components uniformly to obtain the antibody diluent.

[0084] Method for detecting prostate lesion tissue using prostate lesion tissue immunofluorescence detection kit Detection method:

[0085] 1. Sample preparation: 1) Paraffin sections: slice the biopsy tissue provided by the patient after dehydration, transparency, wax immersion, embedding and other operations, and then slice it with a tissue slicer. The biopsy tissue of the prostate cancer patient is provided by the cooperative hospital. Put it into xylene for 15min-xylene for 15min-anhydrous ethanol for 5min-anhydrous ethanol for 5min-95% ethanol for 5min-85% ethanol for 5min-75% ethanol for 5min, and wash it with distilled water. 2) Frozen sections: fix the frozen sections for 10-30min, wash with PBS solution for 5min, repeat 3 times, add 0.3wt% triton-X100 permeabilization solution for 20min, wash with PBS solution for 5min, repeat 3 times. 3) Cell slides or cell smears: fix the cell sample for 20min, wash with PBS solution for 5min, repeat 3 times, add 0.3wt% tritonX-100 permeabilization solution for 20min, wash with PBS solution for 5min, repeat 3 times.

[0086] 2. Antigen repair: Place tissue sections in a container filled with EDTA alkaline antigen repair solution at pH = 9.0 and place in a 95°C water bath for 20 minutes. During this process, excessive evaporation of the buffer should be prevented and the slides should not be dried. After natural cooling, place the slides in a PBS solution and shake on a decolorizing shaker for 3 times, 5 minutes each time. (The repair solution and repair conditions are determined according to the tissue type and antigen type. Antibody elution solution is required for frozen sections and cell samples for antibody elution).

[0087] P504S, repair conditions: pH 9.0 EDTA alkaline antigen repair solution, 95℃ water bath for 20min;

[0088] P63, repair conditions: pH 7.0 citric acid antigen repair solution, 95℃ water bath for 25min;

[0089] CK (HMW), retrieval conditions: pH 7.0 citric acid antigen retrieval solution in a 95°C water bath for 25 min.

[0090] 3. Block endogenous peroxidase: Place the slices in 3wt% hydrogen peroxide solution and incubate at room temperature in the dark for 15 minutes. Place the slides in PBS solution and wash them three times on a decolorizing shaker for 5 minutes each time.

[0091] 4. Non-specific target blocking: After the slices are slightly dried, use a histochemical pen to draw a circle around the tissue (to prevent the antibody from flowing away), and then drop the blocking antibody dilution solution in the circle to evenly cover the tissue, and block at room temperature for 30 minutes.

[0092] The antibody diluent contains various protective agents and preservatives, which can be used to block or dilute the primary antibody. The diluted primary antibody can be stored at four degrees for a long time (it can also be stored for one month at room temperature).

[0093] Incubate with primary antibody: Gently shake off the blocking solution, add primary antibody P504S (mouse origin, 1:2000) diluted with antibody diluent on the slices, and incubate the slices flat in a light-proof humid box at 4°C overnight (add a small amount of water in the humid box to prevent antibody evaporation).

[0094] Incubate with poly-HRP secondary antibody: Place the slide in PBS solution and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add the ready-to-use AFIHC001 poly-HRP goat anti-mouse / rabbit universal secondary antibody of the corresponding species to the primary antibody in the circle to cover the tissue, incubate at room temperature in the dark for 50 minutes, and wash three times with PBS solution.

[0095] 7. TSA fluorescent dye reaction solution reaction: Mix TYR-480Plus fluorescent dye and TSA buffer evenly, add the prepared TSA fluorescent dye reaction solution to the slices to evenly cover the tissue and react at room temperature for 7 minutes, and wash three times with PBS solution (in the preliminary experiment, you can dye for 1 minute to wash off the fluorescent dye and then observe the staining effect under a microscope. If the positive result is weak, continue to add fluorescent dye to enhance the staining intensity until the appropriate intensity is reached before proceeding to the next step).

[0096] 8. Antibody elution: Place the paraffin sections in antigen repair solution in a 37°C water bath for 30 minutes to cover the sample, place at 37°C for 10 minutes, discard the eluent, add an appropriate amount of antibody eluent again to cover the sample and place at 37°C for 10 minutes, discard the eluent, and wash three times with PBS solution, each time for 5 minutes (paraffin sections can be eluted with heat repair or antibody eluent, and cells and ice-cut sections need to be eluted with antibody eluent).

[0097] 9. Repeat steps 3-8 (using TYR-529Plus fluorescent dye and primary antibody P63) for the second round of labeling.

[0098] 10. Repeat steps 3-8 (using TYR-570Plus fluorescent dye and primary antibody CK) for the third round of labeling.

[0099] 11. Dapi counterstaining of cell nuclei: Place the slide in PBS solution and shake on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, add Dapi ready-to-use staining solution in the circle and incubate at room temperature for 10 minutes away from light.

[0100] 12. Sealing: Place the slide in PBS solution and wash on a decolorizing shaker for 3 times, 5 minutes each time. After the slices are slightly dried, seal them with anti-fluorescence quenching sealing medium.

[0101] 13. Microscopic examination and photography: The slices are observed and images are collected under a fluorescence microscope / confocal / multi-channel fluorescence scanner / multi-spectral imaging system. The whole scanned pathological slice is imported into the whole slice image and the MSI high-magnification scan is performed according to the tumor area fixed by the adverse factors. When selecting MSI, one area can be selected for analysis or multiple areas can be selected for analysis at the same time. The collected MSI high-magnification images can be used for statistics with inForm / HALO software. See the results. Figure 1 .

[0102] P504s (marked in green in this experiment) is a prostate cancer-specific marker that is highly expressed in prostate cancer and not expressed in normal prostate tissue. p63 (marked in red in this experiment) and HMW CK (marked in pink in this experiment) mark the nucleus and cytoplasm of prostate basal cells, respectively. Normal prostate glands have two layers of cells (basal cells and glandular epithelial cells), while prostate cancer has no or only a small amount of basal cells.

[0103] Example 2. This example provides an immunofluorescence detection kit for prostate lesion tissue, which is prepared from the following raw materials in parts by weight: 60 parts of TYR-xxxPlus fluorescent dye, 45 parts of TSA Buffer, 40 parts of Polymer-HRP monoclonal antibody mouse / rabbit secondary antibody, 30 parts of antibody, 15 parts of Dapi dye solution, 70 parts of antibody diluent, and 70 parts of peroxidase blocking solution. The antibodies include primary antibody P504S, primary antibody P63, and primary antibody CK, and the mass ratio is 10:15:10.

[0104] The TYR-xxxPlus fluorescent dye includes TYR-570Plus fluorescent dye, TYR-620Plus fluorescent dye, and TYR-690Plus fluorescent dye, with a mass ratio of 1:1:1.

[0105] The antibody diluent is prepared from the following raw materials by weight: 100 parts of PBS buffer, 1 part of bovine serum albumin, 2 parts of trehalose, 40 parts of glycerol, 1 part of Tween-60, and 0.5 parts of the preservative prepared in Preparation Example 2. The preparation method is to mix the components uniformly to obtain the antibody diluent.

[0106] The detection method is the same as that in Example 1. The whole scanned pathological slice is imported into the whole slice image and the MSI high-magnification scan is performed according to the tumor area fixed by the adverse factors. When selecting MSI, one area can be selected for analysis or multiple areas can be selected for analysis at the same time. The collected MSI high-magnification images can be used for statistics with the inForm / HALO software. The results are shown in Figure 2 .

[0107] Example 3. This example provides an immunofluorescence detection kit for prostate lesion tissue, which is prepared from the following raw materials in parts by weight: 50 parts of TYR-xxxPlus fluorescent dye, 40 parts of TSA Buffer, 30 parts of Polymer-HRP monoclonal antibody mouse / rabbit secondary antibody, 22 parts of antibody, 10 parts of Dapi dye solution, 60 parts of antibody diluent, and 60 parts of peroxidase blocking solution. The antibodies include primary antibody P504S, primary antibody P63, and primary antibody CK, and the mass ratio is 10:13:10.

[0108] The TYR-xxxPlus fluorescent dye includes TYR-480Plus fluorescent dye, TYR-570Plus fluorescent dye, and TYR-780Plus fluorescent dye, and the mass ratio is 1:1:1.

[0109] The antibody diluent is prepared from the following raw materials by weight: 90 parts of PBS buffer, 0.7 parts of bovine serum albumin, 1 part of trehalose, 30 parts of glycerol, 0.7 parts of Tween-20, and 0.3 parts of the preservative prepared in Preparation Example 3. The preparation method is to mix the components uniformly to obtain the antibody diluent.

[0110] The detection method is the same as that in Example 1. The whole scanned pathological slice is imported into the whole slice image and the MSI high-magnification scan is performed according to the tumor area fixed by the adverse factors. When selecting MSI, one area can be selected for analysis or multiple areas can be selected for analysis at the same time. The collected MSI high-magnification images can be used for statistics with the inForm / HALO software. The results are shown in Figure 3 .

[0111] Depend on Figure 1-3It can be seen that the staining effect of the kit used in the above different prostate cancer patients is as follows: the three test results all show strong green fluorescence signals and red or pink material signals, and the three-label staining results all show strong positive P504S, negative P63 and CK (HMV). The prostate cancer-specific marker P504s is highly expressed in prostate cancer, and there are no or a small amount of residual basal cells. The test results of the kit of the present invention are consistent with the pathological characteristics of prostate cancer. The test results show that P504s has a strong green fluorescence signal and is highly expressed in prostate cancer; while P63 and CK (HMV) are markers for prostate basal cells, and no expression signals can be detected, and the results are negative.

[0112] Comparative Example 1

[0113] Compared with Example 3, the difference is that the preservative is prepared by Comparative Preparation Example 1. Figure 4 As can be seen from the figure, the background of the staining result is darker and the specificity is not that strong. This method does not have good resolution.

[0114] Comparative Example 2

[0115] Compared with Example 3, the difference is that the preservative is prepared by Comparative Preparation Example 2. Figure 5 As can be seen from the figure, the background of the staining result is darker and the specificity is not that strong. This method does not have good resolution.

[0116] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions, improvements, etc. made within the spirit and principle of the present invention should be included in the protection scope of the present invention.

Claims

1. A prostate lesion tissue immunofluorescence detection kit, characterized in that: The preparation is made from the following raw materials by weight: 40-60 parts of TYR-xxxPlus fluorescent dye, 30-45 parts of TSA Buffer, 20-40 parts of Polymer-HRP monoclonal antibody mouse / rabbit secondary antibody, 15-30 parts of antibody, 5-15 parts of Dapi dye solution, 50-70 parts of antibody diluent, and 50-70 parts of peroxidase blocking solution; The antibody diluent is prepared from the following raw materials by weight: 80-100 parts of PBS buffer, 0.5-1 part of bovine serum albumin, 0.5-2 parts of trehalose, 20-40 parts of glycerol, 0.5-1 part of surfactant, and 0.1-0.5 part of preservative; The preparation method of the preservative is as follows: S1. Preparation of lauric acid vitamin C ester: lauric acid and vitamin C are added to toluene, concentrated sulfuric acid is added, and the reaction mixture is heated to reflux, the reaction solution is washed with saturated sodium carbonate and water in turn, the organic layer is collected, and separated by column chromatography to obtain lauric acid vitamin C ester; the molar ratio of lauric acid to vitamin C is 1:0.9-1; S2. Preparation of carboxylated chitosan: dissolving chitosan in acid solution, adding α-ketoglutaric acid, heating and stirring to react, adding sodium borohydride, stirring to react, adding ethanol to precipitate, washing, and drying to obtain carboxylated chitosan; the mass ratio of chitosan, α-ketoglutaric acid, and sodium borohydride is 10:14-17:3-5; S3. Preparation of preservative: adding carboxylated chitosan and lauric acid vitamin C ester to toluene, adding concentrated sulfuric acid, heating to reflux reaction, adding ethanol to precipitate, washing, and drying to obtain a preservative; the mass ratio of the carboxylated chitosan, lauric acid vitamin C ester and concentrated sulfuric acid is 10:3-4:0.5-1.

2. The prostate lesion tissue immunofluorescence detection kit according to claim 1, characterized in that: The TYR-xxxPlus fluorescent dye is selected from any three of TYR-480Plus fluorescent dye, TYR-520Plus fluorescent dye, TYR-570Plus fluorescent dye, TYR-620Plus fluorescent dye, TYR-690Plus fluorescent dye, and TYR-780Plus fluorescent dye; the antibodies include primary antibody P504S, primary antibody P63, and primary antibody CK, with a mass ratio of 10:12-15:

10.

3. The prostate lesion tissue immunofluorescence detection kit according to claim 1, characterized in that: The surfactant is selected from at least one of Tween-20, Tween-40, Tween-60, Tween-80 and Tween-85.

4. The prostate lesion tissue immunofluorescence detection kit according to claim 1, characterized in that: The amount of concentrated sulfuric acid added in step S1 is 1-2wt% of the total mass of the system, and the heating reflux reaction time is 3-5h; the acid solution in step S2 is 2-3wt% acetic acid or lactic acid, the temperature of the heating and stirring reaction is 35-40°C, the time is 20-24h, and the stirring reaction time is 10-12h; the heating reflux reaction time in step S3 is 4-6h.

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