Immune cell chemical detection kit for identifying urothelium tumor and staining method thereof

Abstract

The invention relates to an immune cell chemical detection kit for identifying urinary tract epithelial tumors and a dyeing method thereof, and belongs to the field of immune detection. The kit comprises 10% neutral buffer formalin, a nuclear primary antibody, a cytoplasm primary antibody, an HRP-labeled secondary antibody polymer, an AP-labeled secondary antibody polymer, a DAB staining solution and a basic fuchsin staining solution. The cell nucleus primary antibody is selected from p53 or Ki67, and the cytoplasm primary antibody is selected from CK17 or CK20. According to the method, urine exfoliated cells are subjected to liquid-based slide making and combined with an immunochemical double staining method to identify the urinary tract epithelium tumor related antigen, and during immunochemical double staining, a 10% neutral buffer formalin short-time fixation and antigen low-temperature repair method is utilized, so that the integrity of the cells is ensured, the identification time is greatly saved, and the identification efficiency is improved. In addition, the antigen is fully exposed, clearer positive cells are obtained, the dyeing quality is ensured, and the identification accuracy of the urothelium tumor associated antigen is improved.

Description

Technical Field

[0001] The invention relates to an immunocytochemical detection kit and a staining method thereof for identifying urothelial tumors, and belongs to the field of immunodetection. Background Art

[0002] Urothelial carcinoma (UC) is a urinary system disease, which is divided into upper urinary tract urothelial carcinoma and lower urinary tract urothelial carcinoma, mainly including renal pelvic tumors, fallopian tube tumors, bladder tumors, urethral tumors, etc., with high incidence, multifocality, and easy recurrence. Bladder tumors have the highest incidence among urothelial tumors, with an incidence of up to 90%. Currently, cystoscopy and combined pathological biopsy technology are the gold standard for diagnosing primary and recurrent bladder cancer. Because cystoscopy is an invasive examination, it has the disadvantages of poor comfort, low patient compliance, easy to cause complications such as bleeding and infection, and high price. It also has poor sensitivity to carcinoma in situ and is difficult to meet clinical needs, thus missing the opportunity for early diagnosis and increasing the risk of disease. Urine exfoliated cytology is a detection technology that has emerged in recent years. By observing and examining the exfoliated cells in the urine, it can determine whether there is urothelial carcinoma or potential precancerous lesions, which to a certain extent makes up for the shortcomings of microscopy. However, there are fewer exfoliated epithelial cells in urine, which are prone to degeneration or autolysis, and often cannot be clearly diagnosed. This places high demands on diagnostic physicians and is difficult to meet clinical needs. Therefore, it is very necessary to provide a non-invasive, rapid, small sample volume, and high accuracy method for detecting urothelial tumors.

[0003] Immunochemical staining is a commonly used technical method in histopathology. It can use the principle of specific binding of antigen and antibody to detect whether the target antigen exists in cells or tissues and determine the protein expression in tumor cells or tissues. It is the gold standard for tumor pathology diagnosis. Immunochemical staining is highly specific, sensitive, simple, fast and low-cost, which is conducive to in-depth research in the field of pathology.

[0004] Immunochemical double staining is one of the most commonly used immunocytochemical staining methods and is often used as an effective tool for identifying tumors, especially urothelial tumors. It uses two primary antibodies of different species, two secondary antibodies with different reaction systems, and different color development systems. It can simultaneously detect the expression of two antigens in one section to discover their localization, morphology, and functional relationships.

[0005] The common preparation techniques for immunocytochemical staining are mainly cell wax block sectioning and liquid-based preparation. Cell wax block sectioning is a traditional preparation method with wide application. Its production process includes sampling, fixation, washing and dehydration, transparency, wax immersion, embedding, sectioning and other steps. The process is relatively cumbersome and time-consuming. It usually takes 3 to 5 days to get the results, which greatly increases the identification time. Liquid-based cytology technology has simple procedures and is easy to operate. It has great advantages in automation and standardization. In addition, liquid-based preparation cells are fixed in time and the morphology is better preserved, which is conducive to subsequent identification and significantly improves the accuracy and sensitivity of identification.

[0006] In the process of identifying urothelial tumors using liquid-based preparations combined with immunochemical double staining, the original state of the cells in liquid-based preparations is highly retained, while immunochemical double staining requires staining of two antigens, and the reagents and staining steps used are relatively large, which can easily result in unsatisfactory staining effects on urine exfoliated cells. For example, the number of positive cells identified is small, the staining is weak, the cell morphology is incomplete, the nuclear details and the nuclear membrane outline are blurred, and these problems affect the identification of urothelial tumor-related antigens. Summary of the invention

[0007] The first purpose of the present invention is to provide an immunocytochemical detection kit that can be used to identify urothelial tumors, so as to solve the problem that the positive rate, staining intensity and staining details need to be improved when performing liquid-based preparation and immunochemical double staining of urine exfoliated cells.

[0008] The second object of the present invention is to provide an immunocytochemical staining method for non-diagnostic purposes to solve the above-mentioned problems.

[0009] To achieve the above first purpose, the technical solution adopted by the present invention is:

[0010] An immunocytochemical detection kit for identifying urothelial tumors, the kit comprising 10% neutral buffered formalin, nuclear primary antibody and cytoplasmic primary antibody, HRP-labeled secondary antibody polymer and AP-labeled secondary antibody polymer, DAB staining solution, basic fuchsin staining solution; the nuclear primary antibody is selected from p53 or Ki67, and the cytoplasmic primary antibody is selected from CK17 or CK20.

[0011] The present invention belongs to the type of improved invention creation. The fixing liquid used in the traditional liquid-based film preparation is generally 95% ethanol. In the present invention, 10% neutral buffered formalin is selected as the fixing liquid. Because 95% ethanol has strong dehydration, it is easy to cause nuclear protein precipitation, affecting the staining of nuclear proteins such as Ki67 and p53; while 10% neutral buffered formalin can form cross-linking between molecules, thereby affecting the configuration of the protein and fixing it, so that the morphological structure of the cell is well preserved. Using it to fix urine exfoliated cells can make urine exfoliated cells show a higher positive rate and a stronger staining effect, and negative cells have clearer nuclear details and nuclear membrane contours.

[0012] The nuclear primary antibody is selected from p53 or Ki67, and the cytoplasmic primary antibody is selected from CK17 or CK20. Immunochemical double staining can simultaneously identify antigens in the nucleus and cytoplasm. The expression of two antigens can be observed in one section and the interaction between two target proteins can be identified.

[0013] The polymer based on HRP labeled in the secondary antibody polymer binds to the antibody and is colored brown by DAB, thereby locating the primary antibody in the nucleus or cytoplasm; the polymer based on AP labeled in the secondary antibody polymer binds to the antibody and is colored red by alkaline fuchsin staining solution, thereby locating the primary antibody in the cytoplasm or nucleus.

[0014] Preferably, the nuclear primary antibody and the cytoplasmic primary antibody are combined primary antibody reagents containing both nuclear primary antibody and cytoplasmic primary antibody; the HRP-labeled secondary antibody polymer and the AP-labeled secondary antibody polymer are combined secondary antibody reagents containing both HRP-labeled secondary antibody polymer and AP-labeled secondary antibody polymer. By using the combined primary antibody reagent and the combined secondary antibody reagent, the double staining process only requires a single primary antibody incubation and a single secondary antibody incubation, which is more time-saving than the sequential double staining method, and can reduce the risk of urine exfoliated cell loss caused by multiple washings with washing buffer.

[0015] Preferably, the HRP-labeled secondary antibody polymer localizes the nuclear primary antibody, and the AP-labeled secondary antibody polymer localizes the cytoplasmic primary antibody.

[0016] Preferably, the kit further comprises urine exfoliated cell preservation solution, peroxidase blocking agent and hematoxylin counterstaining solution, wherein the peroxidase blocking agent can block the endogenous peroxidase of the tissue, reduce the activity of these enzymes, thereby reducing the staining background and making the staining result clearer.

[0017] To achieve the above second purpose, the technical solution adopted by the present invention is:

[0018] An immunocytochemical staining method for non-diagnostic purposes comprises the following steps: sedimentation preparation, 10% neutral buffered formalin fixation, antigen repair, double staining with a nuclear primary antibody and a cytoplasmic primary antibody, wherein the nuclear primary antibody is selected from p53 or Ki67, and the cytoplasmic primary antibody is selected from CK17 or CK20.

[0019] The present invention uses a liquid-based preparation of urine exfoliated cells and combines it with an immunochemical double staining method to identify urothelial tumor-associated antigens. Sedimentation preparation is a major preparation method for liquid-based preparation, which utilizes the natural sedimentation of cells by gravity, does not destroy the morphology and three-dimensional structure of the cells, and the cells are fixed in time, and the morphology is better preserved, which is conducive to the subsequent identification of urothelial tumor-associated antigens. A combination of primary antibodies is selected to recognize antigens in the nucleus and cytoplasm, thereby achieving immunochemical double staining, and the expression of the two antigens can be observed in one slice and the interaction between the two target proteins can be identified.

[0020] Preferably, the 10% neutral buffered formalin fixation time is 5 to 20 minutes.

[0021] More preferably, the 10% neutral buffered formalin fixation time is 5 to 10 minutes.

[0022] Preferably, the antigen repair temperature is 60-90°C and the time is 5-20 minutes. The double staining experiment results show that high temperature easily destroys the cytoplasmic structure, while the low-temperature antigen repair condition can fully expose the antigen, make the cell morphology more complete, and have more cells, ultimately showing a higher positive rate.

[0023] Preferably, the double staining is a combination staining, including using a combined primary antibody reagent containing both a nuclear primary antibody and a cytoplasmic primary antibody for primary antibody incubation, then using a combined secondary antibody reagent containing both an HRP-labeled secondary antibody polymer and an AP-labeled secondary antibody polymer for secondary antibody incubation, and then performing DAB color development and AP color development in sequence.

[0024] Preferably, the double staining is sequential staining, including first incubating with a cytoplasmic primary antibody or a nuclear primary antibody and a corresponding secondary antibody, then incubating with a nuclear primary antibody or a cytoplasmic primary antibody and a corresponding secondary antibody, and then performing DAB color development and AP color development in sequence. BRIEF DESCRIPTION OF THE DRAWINGS

[0025] Figure 1 This is an immunochemical double staining image of identifying urothelial tumor-related antigens using CK17 / Ki67 combination primary antibody as a target in Example 2 of the present invention;

[0026] Figure 2 This is an immunochemical double staining image of identifying urothelial tumor-related antigens using the CK20 / p53 combination primary antibody as a target in Example 3 of the present invention;

[0027] Figure 3 The immunochemical double staining image of urine exfoliated cells fixed with 10% neutral buffered formalin and using CK17 / Ki67 combination primary antibody as a target to identify urothelial tumor-related antigens in Experimental Example 1 of the present invention;

[0028] Figure 4 This is an immunochemical double staining image of urine exfoliated cells fixed with 95% ethanol in Experimental Example 1 of the present invention, and using CK17 / Ki67 combination primary antibody as a target to identify urothelial tumor-related antigens;

[0029] Figure 5 The immunochemical double staining image of the present invention in Experimental Example 2 was obtained by heat repairing with an antigen repair solution at a temperature of 70° C. for 18 minutes and using a CK20 / Ki67 combination primary antibody as a target to identify urothelial tumor-related antigens;

[0030] Figure 6 The immunochemical double staining image of the present invention in Experimental Example 2 was performed by heat repairing with an antigen repair solution at a temperature of 100° C. for 10 minutes, and using the CK20 / Ki67 combination primary antibody as a target to identify urothelial tumor-related antigens;

[0031] Figure 7 This is an immunochemical double staining image of identifying urothelial tumor-related antigens using CK17 / p53 combination primary antibody as a target in Experimental Example 3 of the present invention;

[0032] Figure 8 This is an immunochemical single staining image of identifying urothelial tumor-related antigens using CK17 primary antibody as a target in Experimental Example 3 of the present invention;

[0033] Fig. 9 This is an immunochemical single staining image of identifying urothelial tumor-related antigens using p53 primary antibody as the target in Experimental Example 3 of the present invention. DETAILED DESCRIPTION

[0034] The technical concept of the present invention is to perform liquid-based preparation on urine exfoliated cells, and then realize immunocytochemical double staining by improving the fixation method, antigen repair conditions and selecting appropriate targets. Among them, the use of 10% neutral buffered formalin for fixation can reduce the effect of the fixative on the above-mentioned staining targets; further, by short-term (as short as 5 minutes) 10% neutral buffered formalin fixation, combined with subsequent low-temperature repair of antigens (not more than 90°C), not only the integrity of cell morphology is guaranteed, but also the antigen sites are fully exposed, which is conducive to obtaining clearer positive cells, ensuring the staining quality, and improving the accuracy of identification of urothelial tumor-related antigens. At the same time, relative to urine samples with less cell quantity, this scheme greatly reduces the requirements for cell quantity.

[0035] In order to further reduce the time of immunochemical double staining, reduce the number of buffer washes and reduce cell loss, it is preferred to use a double staining strategy of combined primary antibodies and combined secondary antibodies. Combination primary antibodies such as CK17 / Ki67, CK20 / p53, CK17 / p53, CK20 / Ki67 and corresponding combination secondary antibodies have clear localization of the nucleus and cytoplasm, clear target signals, and are easy to distinguish between cells expressing two antigens at the same time and cells expressing only one antigen. In the later stage, according to the number of double positive cells, auxiliary cytology can be used to analyze the source of urothelial tumor cells, which is conducive to the classification of urothelial carcinoma.

[0036] The specific operation of the method based on liquid-based preparation combined with immunochemical double staining is as follows:

[0037] (1) Cell preservation: Centrifuge urine (1000-2000 rpm, 5-10 min), slowly discard the supernatant, resuspend the urine sediment with PBS buffer, centrifuge again (2000 rpm, 5-10 min), slowly discard the supernatant, and add an appropriate amount of urine exfoliated cell preservation solution to the washed urine sediment;

[0038] (2) Specimen preparation: After the sample is placed in urine exfoliated cell preservation solution for more than 30 minutes, the sample can be prepared by sedimentation preparation method. The sample sedimentation time is 10 to 15 minutes.

[0039] (3) Fixation: Fix with 10% neutral buffered formalin for 5-20 min and wash twice with washing buffer;

[0040] (4) Antigen repair: discard the washing buffer, use a heating device to heat the antigen repair solution to 60-90°C, heat repair the specimen for 5-20 minutes, discard the antigen repair solution after cooling, and wash twice with the washing buffer;

[0041] (5) Peroxidase blocking: Add peroxidase blocking agent, incubate at room temperature for 3-5 min, and then wash twice with washing buffer;

[0042] (6) Primary antibody incubation: Add CK17 / Ki67, CK20 / p53, CK17 / p53, CK20 / Ki67 combination primary antibodies, incubate at room temperature for 20-60 min, and wash twice with washing buffer;

[0043] (7) Secondary antibody incubation: discard the washing buffer, add the combined secondary antibody, incubate at room temperature for 10 to 40 min, and wash with the washing buffer;

[0044] (8) DAB staining: discard the washing buffer, add DAB staining solution, incubate at room temperature for 3-8 min, and wash twice with washing buffer;

[0045] (9) AP staining: discard the washing buffer, add basic fuchsin staining solution, incubate at room temperature for 3-8 min, and wash twice with pure water;

[0046] (10) Re-staining: discard the pure water, re-stain with hematoxylin for 30-60 seconds, and rinse twice with pure water;

[0047] (11) Seal the sections with aqueous mounting medium.

[0048] The main improvement of the present invention is to improve the fixation method and antigen retrieval conditions for liquid-based preparation of urine exfoliated cells. The reagents used, such as nuclear primary antibody and cytoplasmic primary antibody, HRP-labeled secondary antibody polymer and AP-labeled secondary antibody polymer, are all mature products or mature technologies in the field of urothelial tumor diagnosis. The combination of primary antibody reagents and combination of secondary antibody reagents mainly combines two primary antibodies or secondary antibodies into the same reagent to simplify the primary antibody and secondary antibody incubation operations. The relevant reagents can be made of mature products or conventional technologies in the field, which does not affect the implementation of the present invention and the realization of the purpose of the invention.

[0049] The implementation process of the present invention is described in detail below in conjunction with specific embodiments.

[0050] 1. Specific embodiments of an immunocytochemical detection kit for identifying urothelial tumors of the present invention

[0051] Example 1

[0052] The immunocytochemical detection kit for identifying urothelial carcinoma in this embodiment contains the following reagents:

[0053] Urine exfoliated cell preservation solution, 10% neutral buffered formalin, peroxidase blocking agent, combination of primary antibodies, combination of secondary antibodies, DAB staining solution, basic fuchsin staining solution, hematoxylin counterstaining solution, and washing buffer.

[0054] The combination primary antibody is CK17 / Ki67, Ki67 is the nuclear primary antibody, and CK17 is the cytoplasmic primary antibody.

[0055] The combined secondary antibody is a cocktail mixed secondary antibody with high sensitivity; the anti-mouse polymer labeled with HRP in the secondary antibody polymer binds to the mouse anti-human antibody, and is colored brown by DAB to locate Ki67; at the same time, the anti-rabbit polymer labeled with AP in the secondary antibody polymer binds to the rabbit anti-human antibody, and is colored red by alkaline fuchsin staining solution to locate CK17.

[0056] In other implementation situations, the combination of primary antibodies can be selected from CK20 / p53, CK17 / p53, CK20 / Ki67, and the corresponding combination of secondary antibodies is selected based on the above combination of primary antibodies.

[0057] 2. Specific embodiment of the immunocytochemical staining method for non-diagnostic purposes of the present invention

[0058] Example 2

[0059] The immunocytochemical staining method for non-diagnostic purposes in this example uses CK17 / Ki67 as a target to identify urothelial tumor-related antigens. The specific operation is as follows:

[0060] (1) Cell preservation: centrifuge urine (2000 rpm, 7 min), slowly discard the supernatant, resuspend the urine sediment with PBS buffer, centrifuge again (2000 rpm, 7 min), slowly discard the supernatant, and add an appropriate amount of urine exfoliated cell preservation solution (conventional cell preservation solution for liquid-based preparation; Henan Synotech Biotechnology Co., Ltd., cell preservation solution, product number: TC1001) to the washed urine sediment;

[0061] (2) Specimen preparation: After the sample is placed in the urine exfoliated cell preservation solution for more than 30 minutes, the sample can be prepared by the sedimentation method. The sample sedimentation time is 10 minutes.

[0062] (3) Fixation: Fix with 10% neutral buffered formalin for 5 min and wash twice with washing buffer;

[0063] (4) Antigen repair: discard the washing buffer, use a heating device to heat the antigen repair solution to 60°C, heat repair the specimen for 20 minutes, discard the antigen repair solution after cooling, and wash twice with the washing buffer;

[0064] (5) Peroxidase blocking: add peroxidase blocking agent, incubate at room temperature for 3 min, and then wash twice with washing buffer;

[0065] (6) Primary antibody incubation: Add CK17 / Ki67 combination primary antibody, incubate at room temperature for 20 min, and wash twice with washing buffer;

[0066] (7) Secondary antibody incubation: discard the washing buffer, add the combined secondary antibody, incubate at room temperature for 40 min, and wash with the washing buffer;

[0067] (8) DAB staining: discard the washing buffer, add DAB staining solution (DAB substrate: buffer = 1:20, prepared immediately; Henan Synotech Biotechnology Co., Ltd.), incubate at room temperature for 8 min, and wash twice with washing buffer;

[0068] (9) AP staining: discard the washing buffer, add alkaline fuchsin staining solution (after the fast red color developer and activator are mixed and reacted for 3 minutes, add the fast red buffer solution, which is prepared and used immediately; Henan Synotech Biotechnology Co., Ltd.), incubate at room temperature for 5 minutes, and wash twice with pure water;

[0069] (10) Restaining: discard the pure water, restain with hematoxylin for 40 seconds, and rinse twice with pure water;

[0070] (11) Seal the sections with aqueous mounting medium.

[0071] The results of immunochemical staining showed that CK17 / Ki67 was used as the target to identify urothelial tumor-related antigens. The nucleus was brown for Ki67 and the cytoplasm was red for CK17 ( Figure 1 , 400 times), it can be seen that the CK17 / Ki67 combination primary antibody is used as the target, with clear localization in the nucleus and cytoplasm, clear target signal, and it is easy to distinguish cells expressing both antigens from cells expressing only one antigen.

[0072] Example 3

[0073] The immunocytochemical staining method for non-diagnostic purposes in this example uses CK20 / p53 as a target to identify urothelial tumor-related antigens. The specific operation is as follows:

[0074] (1) Cell preservation: Urine was centrifuged (2000 rpm, 7 min), the supernatant was slowly discarded, the urine sediment was resuspended with PBS buffer, and centrifuged again (2000 rpm, 7 min), the supernatant was slowly discarded, and an appropriate amount of urine exfoliated cell preservation solution was added to the washed urine sediment;

[0075] (2) Specimen preparation: After the sample is placed in the urine exfoliated cell preservation solution for more than 30 minutes, the sample can be prepared by the sedimentation method. The sample sedimentation time is 10 minutes.

[0076] (3) Fixation: Fix with 10% neutral buffered formalin for 5 min and wash twice with washing buffer;

[0077] (4) Antigen repair: discard the washing buffer, use a heating device to heat the antigen repair solution to 60°C, heat repair the specimen for 20 minutes, discard the antigen repair solution after cooling, and wash twice with the washing buffer;

[0078] (5) Peroxidase blocking: add peroxidase blocking agent, incubate at room temperature for 3 min, and then wash twice with washing buffer;

[0079] (6) Primary antibody incubation: Add CK20 / p53 combination primary antibody, incubate at room temperature for 20 min, and wash twice with washing buffer;

[0080] (7) Secondary antibody incubation: discard the washing buffer, add the combined secondary antibody, incubate at room temperature for 40 min, and wash with the washing buffer;

[0081] (8) DAB staining: discard the washing buffer, add DAB staining solution, incubate at room temperature for 8 min, and wash twice with washing buffer;

[0082] (9) AP staining: discard the washing buffer, add basic fuchsin staining solution, incubate at room temperature for 5 min, and wash twice with pure water;

[0083] (10) Restaining: discard the pure water, restain with hematoxylin for 40 seconds, and rinse twice with pure water;

[0084] (11) Seal the sections with aqueous mounting medium.

[0085] The results of immunochemical staining showed that CK20 / p53 was used as the target to identify urothelial tumor-related antigens, and the nucleus was found to be brown for p53 and the cytoplasm was red for CK20 ( Figure 2 , 400 times), it can be seen that the CK20 / p53 combination primary antibody is used as the target, and the nucleus and cytoplasm are clearly localized, the target signal is clear, and it is easy to distinguish cells expressing both antigens from cells expressing only one antigen.

[0086] 3. Experimental Examples

[0087] Experimental Example 1 Staining Effects of Different Fixation Methods

[0088] This experimental example explores the staining effects of different fixation methods. The specific operations are as follows:

[0089] (1) Cell preservation: centrifuge urine (1500 rpm, 5 min), slowly discard the supernatant, resuspend the urine sediment with PBS buffer, centrifuge again (1500 rpm, 5 min), slowly discard the supernatant, and add an appropriate amount of urine exfoliated cell preservation solution to the washed urine sediment;

[0090] (2) Specimen preparation: After the sample is placed in the urine exfoliated cell preservation solution for more than 60 minutes, the sample can be prepared by the sedimentation method. The sample sedimentation time is 10 minutes.

[0091] (3) Fixation: The two specimens were fixed with 10% neutral buffered formalin for 10 min and 95% ethanol for 5 min, respectively, and washed twice with washing solution;

[0092] (4) Antigen repair: discard the washing buffer, use a heating device to heat the antigen repair solution to 90°C, heat repair the specimen for 5 minutes, discard the antigen repair solution after cooling, and wash twice with the washing buffer;

[0093] (5) Peroxidase blocking: add peroxidase blocking agent, incubate at room temperature for 4 min, and then wash twice with washing buffer;

[0094] (6) Primary antibody incubation: Add CK17 / Ki67 combination primary antibody, incubate at room temperature for 60 min, and wash twice with washing buffer;

[0095] (7) Secondary antibody incubation: discard the washing buffer, add the combined secondary antibody, incubate at room temperature for 10 min, and wash with the washing buffer;

[0096] (8) DAB staining: discard the washing buffer, add DAB staining solution, incubate at room temperature for 4 min, and wash twice with washing buffer;

[0097] (9) AP staining: discard the washing buffer, add basic fuchsin staining solution, incubate at room temperature for 4 min, and wash twice with pure water;

[0098] (10) Restaining: discard the pure water, restain with hematoxylin for 30 seconds, and rinse twice with pure water;

[0099] (11) Seal the sections with aqueous mounting medium.

[0100] The results of immunochemical staining showed that compared with the urine exfoliated cells fixed with 95% ethanol ( Figure 4 , 400 times), and fixed urine exfoliated cells with 10% neutral buffered formalin, Ki67 showed a higher positive rate and stronger staining intensity, and in the specimens fixed with 10% neutral buffered formalin, Ki67-negative cells had clearer nuclear details and nuclear membrane contours ( Figure 3 , 400 times). Among them, the volume of the nucleus of urine exfoliated cells was significantly increased when 95% ethanol was used to fix the urine exfoliated cells, and the Ki67 antigen located in the nucleus was lost. The positive rate was lower than that of 10% neutral buffered formalin fixed urine exfoliated cells ( Figure 3 , 400 times). Therefore, the use of 10% neutral buffered formalin can improve the accuracy of identifying urothelial tumor-related antigens and reduce the occurrence of false negatives.

[0101] Experimental Example 2 Staining effects of different antigen retrieval conditions

[0102] This experiment explores the staining effects of different antigen retrieval conditions. The specific operations are as follows:

[0103] (1) Cell preservation: Centrifuge urine (1000 rpm, 10 min), slowly discard the supernatant, resuspend the urine sediment with PBS buffer, centrifuge again (1000 rpm, 10 min), slowly discard the supernatant, and add an appropriate amount of urine exfoliated cell preservation solution to the washed urine sediment;

[0104] (2) Specimen preparation: After the sample is placed in the urine exfoliated cell preservation solution for more than 90 minutes, the sample can be prepared by the sedimentation method. The sample sedimentation time is 15 minutes.

[0105] (3) Fixation: Fix with 10% neutral buffered formalin for 10 min and wash twice with washing solution;

[0106] (4) Antigen repair: discard the cleaning solution, heat the antigen repair solution to 70°C for one specimen using a heating device, heat repair for 18 min, discard the antigen repair solution after cooling, and wash twice with the cleaning solution; heat the antigen repair solution to 100°C for another specimen using a heating device, heat repair for 10 min, discard the antigen repair solution after cooling, and wash twice with the cleaning solution;

[0107] (5) Peroxidase blocking: add peroxidase blocking agent, incubate at room temperature for 5 min, and then wash twice with washing buffer;

[0108] (6) Primary antibody incubation: Add CK20 / Ki67 combination primary antibody, incubate at room temperature for 30 min, and wash twice with washing buffer;

[0109] (7) Secondary antibody incubation: discard the washing buffer, add the combined secondary antibody, incubate at room temperature for 30 min, and wash with the washing buffer;

[0110] (8) DAB staining: discard the washing buffer, add DAB staining solution, incubate at room temperature for 7 min, and wash twice with washing buffer;

[0111] (9) AP staining: discard the washing buffer, add basic fuchsin staining solution, incubate at room temperature for 8 min, and wash twice with pure water;

[0112] (10) Restaining: discard the pure water, restain with hematoxylin for 50 seconds, and rinse twice with pure water;

[0113] (11) Seal the sections with aqueous mounting medium.

[0114] The results of immunochemical staining showed that the repair condition was 70℃ for 18 min ( Figure 5 , 400 times) compared to 100℃ repair for 10min ( Figure 6 , 400 times), the former has more complete cell morphology and more cells. This shows that high temperature repair can easily destroy the cytoplasmic structure, and the CK20 localization of the former using the low temperature repair strategy shows a higher positive rate.

[0115] Experimental Example 3: Staining Effects of Double Immunostaining and Single Staining of Urine Exfoliated Cells

[0116] This experiment explores the staining effects of urine exfoliated cells double staining and single staining. The specific operations are as follows:

[0117] (1) Cell preservation: Centrifuge urine (1500 rpm, 6 min), slowly discard the supernatant, resuspend the urine sediment with PBS buffer, centrifuge again (1500 rpm, 6 min), slowly discard the supernatant, and add an appropriate amount of urine exfoliated cell preservation solution to the washed urine sediment;

[0118] (2) Specimen preparation: After the sample is placed in the urine exfoliated cell preservation solution for more than 120 minutes, the sample can be prepared by the sedimentation method. The sample sedimentation time is 15 minutes.

[0119] (3) Fixation: Fix with 10% neutral buffered formalin for 10 min and wash twice with washing buffer;

[0120] (4) Antigen repair: discard the washing buffer, use a heating device to heat the antigen repair solution to 80°C, heat repair the specimen for 8 minutes, discard the antigen repair solution after cooling, and wash twice with the washing buffer;

[0121] (5) Peroxidase blocking: add peroxidase blocking agent, incubate at room temperature for 5 min, and then wash twice with washing buffer;

[0122] (6) Primary antibody incubation: Add CK17 / p53 combination primary antibody to double-stained specimens, incubate at room temperature for 40 min, and wash twice with washing buffer; add CK17 and p53 primary antibodies to two single-stained specimens, incubate at room temperature for 40 min, and wash twice with washing buffer;

[0123] (7) Secondary antibody incubation: discard the washing buffer, add the combined secondary antibody to the double-stained specimens, incubate at room temperature for 20 min, and wash with the washing buffer; add HRP-labeled goat anti-mouse / rabbit polymer to the two single-stained specimens, incubate at room temperature for 20 min, and wash with the washing buffer;

[0124] (8) DAB staining: discard the washing buffer, add DAB staining solution, incubate at room temperature for 8 min, and wash twice with washing buffer;

[0125] (9) AP staining: discard the washing buffer, add alkaline fuchsin staining solution, incubate at room temperature for 6 min, and wash twice with pure water; this step can be omitted for single-staining specimens;

[0126] (10) Restaining: discard the pure water, restain with hematoxylin for 60 seconds, and rinse twice with pure water;

[0127] (11) Seal the sections with aqueous mounting medium.

[0128] The results of immunochemical staining show that through immunochemical double staining, only one section needs to be prepared to identify two antigens, identify the expression of the two antigens and observe the interaction between the two proteins ( Figure 7, 400 times), while single staining requires the preparation of two sections, resulting in a decrease in the number of cells per section and the inability to observe the interaction between the two proteins ( Figure 8-Figure 9 , 400 times).

[0129] Experimental Example 4 Staining method of urine exfoliated cells immunostaining

[0130] This experiment explores the difference in staining time between urine exfoliated cell double immunostaining and sequential staining. The specific operation is as follows:

[0131] (1) Cell preservation: Centrifuge urine (1500 rpm, 6 min), slowly discard the supernatant, resuspend the urine sediment with PBS buffer, centrifuge again (1500 rpm, 6 min), slowly discard the supernatant, and add an appropriate amount of urine exfoliated cell preservation solution to the washed urine sediment;

[0132] (2) Specimen preparation: After the sample is placed in the urine exfoliated cell preservation solution for more than 110 minutes, the sample can be prepared by the sedimentation method. The sample sedimentation time is 20 minutes.

[0133] (3) Fixation: Fix with 10% neutral buffered formalin for 10 min and wash twice with washing buffer;

[0134] (4) Antigen repair: discard the washing buffer, use a heating device to heat the antigen repair solution to 80°C, heat repair the specimen for 8 minutes, discard the antigen repair solution after cooling, and wash twice with the washing buffer;

[0135] (5) Peroxidase blocking: add peroxidase blocking agent, incubate at room temperature for 5 min, and then wash twice with washing buffer;

[0136] (6) Primary antibody incubation: Add CK17 primary antibody, incubate at room temperature for 45 min, and wash twice with washing buffer;

[0137] (7) Secondary antibody incubation: discard the washing buffer, add AP-labeled goat anti-rabbit polymer, incubate at room temperature for 25 min, and wash twice with washing buffer;

[0138] (8) Primary antibody incubation: discard the washing buffer, add p53 primary antibody, incubate at room temperature for 35 min, and wash twice with washing buffer;

[0139] (9) Secondary antibody incubation: discard the washing buffer, add HRP-labeled goat anti-mouse polymer, incubate at room temperature for 25 min, and wash twice with washing buffer;

[0140] (10) DAB staining: discard the washing buffer, add DAB staining solution, incubate at room temperature for 8 min, and wash twice with washing buffer;

[0141] (11) AP staining: discard the washing buffer, add alkaline fuchsin staining solution, incubate at room temperature for 6 min, and wash twice with pure water; this step can be omitted for single-staining specimens;

[0142] (12) Restaining: discard the pure water, restain with hematoxylin for 60 seconds, and rinse twice with pure water;

[0143] (13) Seal the sections with aqueous mounting medium.

[0144] From the immunochemical procedure, it can be seen that the antibody incubation time of the urine exfoliated cell double staining method is about 60 to 70 minutes, while the antibody incubation time of the urine exfoliated cell double staining method is 130 minutes. The urine exfoliated cell double staining method saves 60 to 70 minutes compared with the sequential staining method, reduces the risk of urine exfoliated cell loss caused by multiple washing with washing buffer and reduces the waiting time for patients.

Claims

1. An immunocytochemical detection kit for identifying urothelial tumors, characterized in that: The kit comprises 10% neutral buffered formalin, nuclear primary antibody and cytoplasmic primary antibody, HRP-labeled secondary antibody polymer and AP-labeled secondary antibody polymer, DAB staining solution and basic fuchsin staining solution; the nuclear primary antibody is selected from p53 or Ki67, and the cytoplasmic primary antibody is selected from CK17 or CK20.

2. The immunocytochemical detection kit for identifying urothelial tumors according to claim 1, characterized in that: The nuclear primary antibody and cytoplasmic primary antibody are combined primary antibody reagents containing both nuclear primary antibody and cytoplasmic primary antibody; the HRP-labeled secondary antibody polymer and AP-labeled secondary antibody polymer are combined secondary antibody reagents containing both HRP-labeled secondary antibody polymer and AP-labeled secondary antibody polymer.

3. The immunocytochemical detection kit for identifying urothelial tumors according to claim 1 or 2, characterized in that: The HRP-labeled secondary antibody polymer localizes the nuclear primary antibody, and the AP-labeled secondary antibody polymer localizes the cytoplasmic primary antibody.

4. The immunocytochemical detection kit for identifying urothelial tumors according to claim 1, characterized in that: The kit also includes urine exfoliated cell preservation solution, peroxidase blocking agent and hematoxylin counterstaining solution.

5. An immunocytochemical staining method for non-diagnostic purposes, characterized in that: The following steps are involved: Sedimentation preparation, 10% neutral buffered formalin fixation, antigen retrieval, double staining with nuclear primary antibody and cytoplasmic primary antibody, wherein the nuclear primary antibody is selected from p53 or Ki67, and the cytoplasmic primary antibody is selected from CK17 or CK20.

6. The non-diagnostic immunocytochemical staining method according to claim 5, characterized in that: The 10% neutral buffered formalin fixation time is 5 to 20 minutes.

7. The non-diagnostic immunocytochemical staining method according to claim 6, characterized in that: The 10% neutral buffered formalin fixation time is 5 to 10 minutes.

8. The non-diagnostic immunocytochemical staining method according to claim 5, characterized in that: The antigen repair temperature is 60-90° C. and the time is 5-20 min.

9. The non-diagnostic immunocytochemical staining method according to claim 5, characterized in that: The double staining is a combination staining, including using a combined primary antibody reagent containing both a nuclear primary antibody and a cytoplasmic primary antibody for primary antibody incubation, then using a combined secondary antibody reagent containing both an HRP-labeled secondary antibody polymer and an AP-labeled secondary antibody polymer for secondary antibody incubation, and then performing DAB color development and AP color development in sequence.

10. The non-diagnostic immunocytochemical staining method according to claim 5, characterized in that: The double staining is sequential staining, including first incubating with a cytoplasmic primary antibody or a nuclear primary antibody and a corresponding secondary antibody, then incubating with a nuclear primary antibody or a cytoplasmic primary antibody and a corresponding secondary antibody, and then performing DAB color development and AP color development in sequence.

Images