CN113281515A - TIPE3 immunohistochemical detection kit and use method and application thereof - Google Patents
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Abstract
The invention provides a TIPE3 immunohistochemical detection kit and a use method and application thereof, belonging to the technical field of tumor detection. The research of the invention finds that the expression of TIPE3 in pancreatic cancer is obviously increased, is closely related to the tumor invasion and metastasis potential and is an independent prognosis risk factor of a pancreatic cancer patient, so that the aim of helping to judge the pancreatic cancer malignancy and the patient prognosis can be achieved by detecting the expression of TIPE3, and the TIPE3 immunohistochemical detection kit is provided, can effectively predict the pancreatic cancer malignancy and the metastasis potential and can be used for carrying out recurrence and metastasis risk grading on the pancreatic cancer patient; meanwhile, the prognosis condition of the patient can be evaluated, so that the survival rate of the pancreatic cancer patient can be improved, and an important scientific basis is provided for preventing and treating the pancreatic cancer clinically, and therefore, the method has good clinical application value and social benefit.
Description
Technical Field
The invention belongs to the technical field of tumor detection, and particularly relates to a TIPE3 immunohistochemical detection kit and a use method and application thereof.
Background
The information in this background section is only for enhancement of understanding of the general background of the invention and is not necessarily to be construed as an admission or any form of suggestion that this information forms the prior art that is already known to a person of ordinary skill in the art.
Pancreatic cancer is one of the most malignant tumors of the digestive system, with hidden onset, rapid progression, extremely poor prognosis, and almost equal mortality. Recent statistics by the American Cancer Society (ACS) show that the expected incidence of pancreatic cancer is second in digestive tumors and that mortality rates are fourth of all malignancies. Pancreatic cancer is expected to become the second largest killer of all malignancies in 2030.
Although the diagnosis and treatment modes of pancreatic cancer are continuously updated, the prognosis of patients is not significantly improved in the last forty years, the survival rate in 5 years is only about 6%, and the patients are malignant tumors with the worst prognosis at present, so that effective combined detection is urgently needed in clinic, and the overall treatment effect is improved. In addition, the malignancy degree of the pancreatic cancer can be judged only according to the stage of the TNM of the pancreatic cancer, and the higher the understanding degree of the pancreatic cancer is, the more beneficial the treatment of the patient is.
The tumor necrosis factor alpha-induced protein 8(tumor necrosis factor alpha-induced protein 8, TNFAIP8 or TIPE) family has a total of 4 members: TIPE, TIPE1, TIPE2 and TIPE3, which have a high degree of structural homology. Wherein TIPE3 was found in 2008 [ see: sun, H., et al, TIPE2, a negative regulator of intake and adaptive immunity that is immune to ammonia, J, cell.2008,133(3): 415. 426.). Related studies have shown that TIPE3 is a transporter of the phosphatidylinositol second messenger, affecting the growth, proliferation, invasion and migration of tumor cells by activating the phosphatidylinositol 3-kinase/protein kinase B (PI3K/AKT) pathway [ Moniz, L., et al.A. new TIPE of phosphoinositide regulator in Cancer [ J ]. Cancer cell 2014,26(4): 443-.
Immunohistochemistry (IHC) detection is a research that utilizes the principle of antigen-antibody specific binding, and uses a chemical reaction to color a color-developing agent (fluorescein, enzyme, metal ion, isotope) of a labeled antibody to determine the antigens (polypeptide and protein) in tissues or cells, and to localize, qualitatively and relatively quantitatively the antigens. For example, the enzyme is linked to the antibody by covalent bond to produce an enzyme-labeled antibody, and then the enzyme is used to specifically catalyze the substrate to produce a colored insoluble product or particles with a certain electron density, and various antigen components are positioned on the surface of the cell or in the cell under a common microscope or electron microscope. Currently, immunohistochemical indirect staining methods are usually used for the tests, and by using this technique, various antigenic substances, such as proteins, polypeptides, enzymes, hormones, pathogens, receptors, etc., can be detected at the tissue level or the cellular level. Since immunohistochemistry has the characteristics of strong specificity, high sensitivity, accurate positioning and the like, and can organically combine morphological research and functional research together, the new technology is widely applied to many fields of biological and medical research. In pathological studies, the role and significance of immunohistochemistry is more important. However, the inventor finds that no report on a method for evaluating the malignancy degree of pancreatic cancer and the prognosis of a patient by using immunohistochemical detection TIPE3 exists at present.
Disclosure of Invention
Based on the defects of the prior art, the invention provides a TIPE3 immunohistochemical detection kit, and a use method and application thereof. The research of the invention finds that the expression of TIPE3 in pancreatic cancer is obviously increased, is closely related to the tumor invasion and metastasis potential, and is an independent prognosis risk factor of a pancreatic cancer patient, so that the aim of helping to judge the malignancy degree of pancreatic cancer and the prognosis of the patient can be achieved by detecting the expression of TIPE3, and the TIPE3 immunohistochemical detection kit is provided, so that the kit has good practical application value.
In a first aspect of the invention, the invention provides an application of TIPE3 as a diagnostic marker in preparation of pancreatic cancer related detection products.
The pancreatic cancer related detection product comprises a TIPE3 immunohistochemical detection kit which can be used for judging the malignancy degree of pancreatic cancer and the prognosis condition of a patient.
In a second aspect of the present invention, there is provided a TIPE3 immunohistochemical detection kit, wherein the TIPE3 immunohistochemical detection kit comprises at least the following components: a first antibody, a second antibody, a reagent for inactivating peroxidase and biotin in a tissue section, a color development reagent for reacting with horseradish peroxidase and developing color, and a reagent for specifically dyeing cell nuclei in tissues;
the primary antibody is specifically a TIPE3 specific antibody prepared against human TIPE3 antigen, such as rabbit anti-human TIPE3 polyclonal antibody, and the like, and is not specifically limited herein.
Correspondingly, the secondary antibody only needs to be matched with the species, class or subclass of the primary antibody, for example, when the primary antibody is a rabbit anti-human TIPE3 polyclonal antibody, the secondary antibody can be goat anti-rabbit IgG (goat anti-rabbit IgG-HRP).
In a third aspect of the present invention, there is provided a method of using the above TIPE3 immunohistochemical detection kit for immunohistochemical staining diagnosis of tissue sections, the method comprising:
s1, fixing the tissue to be detected of the person to be detected to prepare a paraffin slice;
s2, reacting the processed paraffin wax sheet with a primary antibody and a secondary antibody in sequence;
and S3, after the reaction is finished, cleaning the section, and observing the section through a microscope after DAB staining, hematoxylin counterstaining, dehydration and transparent sealing.
In a fourth aspect of the present invention, there is provided the use of the TIPE3 immunohistochemical detection kit and/or the method of use described above in tumor detection.
Wherein the tumor is a pancreatic cancer;
the tumor detection specifically comprises: judging the malignancy degree and the metastatic potential of the pancreatic cancer and the prognosis condition of the patient (including the survival rate of the pancreatic cancer patient).
The beneficial technical effects of one or more technical schemes are as follows:
the technical scheme is based on that the cancer tissue TIPE3 of a pancreatic cancer patient has higher immunohistochemical staining intensity and wider range, and the malignant degree of pancreatic cancer is higher; on the contrary, the degree of malignancy is lower, so that a TIPE3 immunohistochemical detection kit is prepared, the kit can effectively distinguish pancreatic cancer tissues from normal tissues, and a new diagnosis and classification basis can be provided for individualized treatment of pancreatic cancer.
The kit can effectively predict the malignancy degree and the metastatic potential of pancreatic cancer, and carries out recurrence and metastasis risk classification on pancreatic cancer patients; meanwhile, the prognosis condition of the patient can be evaluated, so that the survival rate of the pancreatic cancer patient can be improved, and an important scientific basis is provided for preventing and treating the pancreatic cancer clinically, and therefore, the method has good clinical application value and social benefit.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, are included to provide a further understanding of the invention, and are incorporated in and constitute a part of this specification, illustrate exemplary embodiments of the invention and together with the description serve to explain the invention and not to limit the invention.
FIG. 1 is a survival curve for patients with high and low expression of TIPE3 according to an embodiment of the present invention;
FIG. 2 is an IHC staining pattern of normal tissue surrounding pancreatic cancer in an embodiment of the present invention;
FIG. 3 is an IHC staining pattern of pancreatic cancer tissue in an embodiment of the invention;
FIG. 4 is an IHC staining pattern of pancreatic cancer tissue with negative lymph node metastasis in an embodiment of the invention;
FIG. 5 is an IHC staining pattern of pancreatic cancer tissue with positive lymph node metastasis in an embodiment of the invention;
FIG. 6 is an IHC staining pattern of lymph node tissue where metastasis does not occur in an embodiment of the present invention;
FIG. 7 is an IHC staining pattern of lymph node tissue where metastasis occurs in an embodiment of the invention;
FIG. 8 is a ROC curve for predicting whether a pancreatic cancer patient will develop lymph node metastasis using the present method in an embodiment of the present invention.
Detailed Description
It is to be understood that the following detailed description is exemplary and is intended to provide further explanation of the invention as claimed. Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs.
It is noted that the terminology used herein is for the purpose of describing particular embodiments only and is not intended to be limiting of example embodiments in accordance with the present application. As used herein, the singular forms "a", "an" and "the" are intended to include the plural forms as well, and it should be understood that when the terms "comprises" and/or "comprising" are used in this specification, they specify the presence of stated features, steps, operations, devices, components, and/or combinations thereof, unless the context clearly indicates otherwise.
As mentioned above, no report on the method for assessing the malignancy of pancreatic cancer and the prognosis of patients by using immunohistochemical detection of TIPE3 is available. The inventor finds that the expression of TIPE3 protein in the immunohistochemical staining of pancreatic cancer tissues is obviously higher than that of surrounding non-cancerous tissues by carrying out immunohistochemical detection on the cancer tissues of pancreatic cancer patients through TIPE3 immunohistochemical detection. And the cancer tissue TIPE3 immunohistochemical detection TIPE3 protein of the pancreatic cancer patient with lymph node metastasis is higher than that of the pancreatic cancer patient without lymph node metastasis. Furthermore, the survival rate of pancreatic cancer patients with high TIPE3 expression was lower than that of pancreatic cancer patients with low TIPE3 expression. Therefore, the cancer tissue TIPE3 of the pancreatic cancer patient has higher immunohistochemical staining intensity and wider range, and the malignant degree of the pancreatic cancer is higher; conversely, the less malignant the degree. The present invention has been made based on the above results.
In one embodiment of the invention, the invention provides application of TIPE3 as a diagnostic marker in preparation of pancreatic cancer related detection products.
The pancreatic cancer related detection product comprises a TIPE3 immunohistochemical detection kit which can be used for judging the malignancy degree of pancreatic cancer and the prognosis condition of a patient.
In another embodiment of the present invention, there is provided a TIPE3 immunohistochemical detection kit, wherein the TIPE3 immunohistochemical detection kit comprises at least the following components: a first antibody, a second antibody, a reagent for inactivating peroxidase and biotin in a tissue section, a color development reagent for reacting with horseradish peroxidase and developing color, and a reagent for specifically dyeing cell nuclei in tissues;
the primary antibody is specifically a TIPE3 specific antibody prepared against human TIPE3 antigen, such as rabbit anti-human TIPE3 polyclonal antibody, and the like, and is not specifically limited herein.
Correspondingly, the secondary antibody only needs to be matched with the species, class or subclass of the primary antibody, for example, when the primary antibody is a rabbit anti-human TIPE3 polyclonal antibody, the secondary antibody can be goat anti-rabbit IgG (goat anti-rabbit IgG-HRP).
The agent for inactivating peroxidase and biotin in the tissue section may be 0.3% H2O2And (3) solution.
The color developing reagent which reacts with horseradish peroxidase and develops color can be DBA solution.
The agent that specifically stains nuclei in the tissue may be hematoxylin.
In yet another embodiment of the present invention, the TIPE3 immunohistochemical detection kit may further comprise a fixative, a deparaffinization hydration agent, a serum blocking agent, a buffer, an antibody diluent, a dehydration clearing agent, and the like.
Wherein, the fixing agent can be neutral buffer 4% paraformaldehyde solution, Bouin's solution or mDF solution.
The serum blocking agent is generally an antibody in the serum of the animal itself, from the same source as the secondary antibody, which binds to a site in the tissue to which the secondary antibody binds in advance; calf serum, BSA, sheep serum, etc. may also be used, but not in line with the primary antibody source.
The buffer may be PBS buffer.
The antibody diluent can be PBS, or special antibody diluent which is favorable for recycling the antibody for multiple times and is added with components such as sodium azide preservative, BSA stabilizer and the like.
The dewaxing hydration agent and the dehydration clearing agent are xylene and ethanol (comprising absolute ethanol, 95% ethanol, 85% ethanol and 75% ethanol) only in the completely opposite using sequence.
In another embodiment of the present invention, there is provided a method of using the above TIPE3 immunohistochemical detection kit for immunohistochemical staining diagnosis of tissue sections, the method comprising:
s1, fixing the tissue to be detected of the person to be detected to prepare a paraffin slice;
s2, reacting the processed paraffin wax sheet with a primary antibody and a secondary antibody in sequence;
and S3, after the reaction is finished, cleaning the section, and observing the section through a microscope after DAB staining, hematoxylin counterstaining, dehydration and transparent sealing.
In step S1, the tissue to be tested may be a pancreatic tissue (e.g., a pancreatic cancer tissue).
In step S2, the processing procedure of the paraffin plate includes: dewaxing and hydrating the prepared paraffin wax tablets, performing antigen retrieval, inactivating peroxidase and biotin, and sealing serum.
In the step S3, the microscope observation includes: randomly selecting three fields for each sample, and each group comprises about 500 cells; semi-quantitative scoring (0, negative; 1, +; 2, + +; 3, + ++) based on hematoxylin staining intensity, on a four-point basis (< 1%, 0 min.; 1-25%, 1 min.; 26-50%, 2 min.; 60-75%, 3 min.; 76-100%, 4 min.) based on the percentage of positive cells; the two scores for each sample were combined to give the final TIPE2 expression score (IHC sum score). The cut-off points defining the expression levels are: 0-3 points, low expression; 4-8 points, high expression.
In yet another embodiment of the present invention, the use of the TIPE3 immunohistochemical detection kit and/or methods of use described above in tumor detection is provided.
Wherein the tumor is a pancreatic cancer;
the tumor detection specifically comprises: judging the malignancy degree and the metastatic potential of the pancreatic cancer and the prognosis condition of the patient (including the survival rate of the pancreatic cancer patient).
The invention is further illustrated by the following examples, which are not to be construed as limiting the invention thereto. It should be understood that these examples are for illustrative purposes only and are not intended to limit the scope of the present invention.
Examples
The method for evaluating the malignancy of pancreatic cancer and the prognosis of a patient by using the TIPE3 immunohistochemical detection kit comprises the following five parts of sample treatment, initial test, antibody incubation, color development and conclusion obtained by observation, and is specifically divided into the following steps:
(1) tissue fixation: after a pancreatic cancer sample is taken from a patient, a treatment with a fixative is required in order to maintain the inherent morphology and structure of cells, preserve the antigenicity of tissue cells, remove lipids that interfere with antigen-antibody binding, and facilitate the antigen-antibody binding to obtain a good staining result. Neutral buffered 4% paraformaldehyde solution (Bouin's solution, mDF solution, etc.) is usually used. The tissue fixation time is preferably within l2h, and generally should not exceed 24 hours. Since the intensity of the tissue antigen detected will gradually decrease with the fixed time.
(2) Tabletting: can be used as a paraffin slice, and has the advantages of simple storage, beautiful structure and long storage time. The tissue is first dehydrated with a gradient of ethanol (from low to high, usually starting with 50% ethanol, through 70%, 85%, 95% to pure ethanol), then impregnated with a clearing agent, then completely waxed, and finally sectioned. The paraffin flakes are generally sliced to about 5 μm. And (5) after slicing, carrying out surface mounting (slice fishing) and baking. A thin layer of protein glycerol can be coated on a glass slide, then the wax sheet is flattened in warm water (about 45 ℃), the glass slide is fished out until the glass slide is laid right, the excessive moisture is sucked by filter paper, and the glass slide is put into a 45 ℃ incubator to be dried.
(3) Dewaxing and hydrating paraffin wax tablets: in order for reagents such as antibodies to sufficiently bind to antigens in tissues, it is necessary to wash pancreatic cancer sample sections sequentially with xylene and gradient ethanol (from high to low). Xylene I for 10min and xylene II for 10 min; anhydrous ethanol for 5min, 95% ethanol for 5min, 85% ethanol for 5min, and 75% ethanol for 5 min; washed 2 times with Phosphate Buffered Saline (PBS) and placed on a shaker.
(4) Antigen retrieval: in the process of fixing part of antigens in tissues by formaldehyde or paraformaldehyde, cross-linking among proteins and blocking of aldehyde groups can occur, so that the antigenicity is lost; through antigen retrieval, intracellular antigenic determinants are exposed again, and the antigen detection rate is improved. The common repair methods can be divided into three types from strong to weak, namely high-pressure repair, microwave repair and pancreatin repair. Typically, the tissue slices are placed in a repair solution and then repaired with microwaves for 6min 4 times. And naturally cooling for about 30min after microwave repair.
(5) Inactivating peroxidase and biotin: if the chromogenic system is a peroxidase (HRP) system, or in a conventional ABC processIn the SP method, the immunohistochemical reaction results are easily interfered by endogenous peroxidase and biotin, and in order to prevent the occurrence of nonspecific color development, inactivation by hydrogen peroxide, avidin or the like is required. Moreover, some tissues may have high endogenous peroxidase levels and need to be inactivated. The sample pieces can be put into 0.3% H2O2And (5) incubating for 10 min. And washed twice in PBS for 10min each time.
(6) Serum blocking: serum was blocked to exclude non-specific binding sites and to exclude non-specific staining. Blocking serum is typically an antibody in the serum of the animal itself, from the same source as the secondary antibody, which binds to a site in the tissue that is capable of binding to the secondary antibody in advance; calf serum, BSA, sheep serum, etc. may also be used, but not in line with the primary antibody source. Can be kept at room temperature for 10-30min to prevent over-sealing.
(7) Primary antibody and secondary antibody treatment:
primary antibody incubation temperatures are several: the incubation effect is optimal at 4 ℃, room temperature and 37 ℃, wherein the incubation effect is optimal at 4 ℃; incubation time: this is related to temperature, antibody concentration. The temperature can be 37 ℃ for 1-2h, and the temperature is 4 ℃ for overnight, and then the temperature is rewarming for 45min at 37 ℃ after being taken out from the refrigerator.
The secondary antibody can be incubated for 30min-1h at room temperature or 37 ℃. And the secondary antibody needs to match the species, class or subclass of the primary antibody.
In this example, primary antibodies used for immunohistochemistry were synthesized by wuhan bosch de bioengineering ltd, and the synthesized TIPE3 epitope peptide was designed to have a C-terminal containing 20 amino acids (RPNLKRICEGINKLLDEKVL), and the dilution concentration was 1: 300, incubation time 4 ℃ overnight; the secondary antibody is horseradish peroxidase-labeled goat anti-rabbit IgG (Beijing China fir Jinqiao Biotechnology Co., Ltd.).
The antibody diluent can be PBS, or special antibody diluent which is beneficial to recycling the antibody for multiple times and is added with components such as sodium azide preservative, BSA stabilizer and the like.
(8) Section wash (dip, rinse and rinse): in order to prevent non-specific staining caused by the remaining of reagents such as primary antibody and secondary antibody, it is important to appropriately enhance washing (for a prolonged period and for an increased number of times), and washing before primary antibody incubation is performed: 3min x 3, wash after primary antibody incubation: 5min x 5 times.
(9) DAB color development: and (4) adding a DBA solution dropwise, developing, observing a brown background under an optical microscope, and stopping washing with tap water in real time. The DAB color development time is not fixed, and the color development time is mainly controlled under a microscope, and the washing can be carried out when the specific dyeing is strong and the background coloring is light.
(10) Counterdyeing: hematoxylin counterstaining (nuclear stain) is commonly used. The aim is to form a cell profile and thus better localize the protein of interest. After the dyeing of the piece is finished, washing the piece by running water, then placing the piece in hydrochloric acid alcohol for a plurality of seconds, washing the piece by running water again, and finally placing the piece in ammonia water to turn blue.
(11) Dehydrating a transparent sealing sheet: the dehydration clarification process is the reverse of the dewaxing process. The slides were washed sequentially with graded ethanol (low to high), xylene: 5min of 75% ethanol → 5min of 85% ethanol → 10min of 95% ethanol → 10min of absolute ethanol → 10min of xylene, sucking off the liquid and airing at room temperature. The transparent phase can be xylene II 10min or xylene I10 min. For long-term storage, however, the gel is usually encapsulated with a neutral gum. One can drop a drop of coverslipping solution directly onto the slide tissue and then cover the slide, taking care of the bubble generation. Air drying for half a day.
(12) And (4) observing under a microscope.
Immunohistochemical staining scoring method: blinded assessments were performed independently by two experienced pathologists. When there is a large difference (score difference >2), a third pathologist is required to analyze the results. Under light microscopy, three fields were randomly selected for each sample, with approximately 500 cells per group. Semi-quantitative scores were made according to hematoxylin staining intensity (0, negative; 1, +; 2, + +; 3, + ++), with a four-point score according to the percentage of positive cells (< 1%, 0 points; 1-25%, 1 points; 26-50%, 2 points; 60-75%, 3 points; 76-100%, 4 points). The two scores for each sample were combined to give the final TIPE2 expression score (IHC sum score). The cut-off points defining the expression levels are: 0-3 points, low expression; 4-8 points, high expression.
Cancer tissue TIPE3 immunohistochemical detection is carried out on pancreatic cancer patients, and as shown in figure 3, the expression of TIPE3 protein in immunohistochemical staining of pancreatic cancer tissues is obviously higher than that of surrounding non-cancer tissues (figure 2). In contrast, TIPE3 immunohistochemistry was detected in cancer tissue of pancreatic cancer patients with lymph node metastasis (FIG. 5) and TIPE3 protein expression was higher than in pancreatic cancer patients without lymph node metastasis (FIG. 4). Furthermore, as shown in fig. 1, our clinical data demonstrate that the survival rate of pancreatic cancer patients with high expression of TIPE3 is lower than that of pancreatic cancer patients with low expression of TIPE 3. In addition, lymph node metastasis is more likely to occur with an immunohistochemical staining score of 4 or more. In conclusion, the cancer tissue TIPE3 of the pancreatic cancer patient has higher immunohistochemical staining intensity and wider range, and the malignant degree of pancreatic cancer is higher; conversely, the less malignant the degree.
It should be noted that the above-mentioned embodiments are only preferred embodiments of the present invention, and the present invention is not limited thereto, and although the present invention has been described in detail with reference to the foregoing embodiments, it will be apparent to those skilled in the art that modifications and equivalents can be made in the technical solutions described in the foregoing embodiments, or equivalents thereof. Any modification, equivalent replacement, or improvement made within the spirit and principle of the present invention should be included in the protection scope of the present invention. Although the present invention has been described with reference to the specific embodiments, it should be understood by those skilled in the art that various changes and modifications may be made without departing from the spirit and scope of the invention.
Claims (10)
1. Application of TIPE3 as a diagnostic marker in preparation of pancreatic cancer related detection products.
2. The use of claim 1, wherein the pancreatic cancer-related detection product comprises a TIPE3 immunohistochemical detection kit; the TIPE3 immunohistochemical detection kit is used for judging the pancreatic cancer malignancy degree and the patient prognosis condition.
3. The TIPE3 immunohistochemical detection kit is characterized in that the TIPE3 immunohistochemical detection kit at least comprises the following components: a first antibody, a second antibody, a reagent for inactivating peroxidase and biotin in a tissue section, a color development reagent for reacting with horseradish peroxidase and developing color, and a reagent for specifically dyeing cell nuclei in tissues;
wherein the primary antibody is a TIPE3 specific antibody specifically prepared against human TIPE3 antigen.
4. The kit of claim 3, wherein the agent that inactivates peroxidase and biotin in the tissue section is H2O2A solution;
the color developing reagent which reacts with the horseradish peroxidase and develops color is a DBA solution;
the reagent for specifically staining the cell nucleus in the tissue is hematoxylin.
5. The kit of claim 3, wherein the TIPE3 immunohistochemical detection kit further comprises a fixative, a deparaffinization hydration agent, a serum blocking agent, a buffer, an antibody diluent, and a dehydration clearing agent.
6. The kit of claim 5, wherein the fixative is neutral buffered 4% paraformaldehyde solution, Bouin's solution, or mDF solution;
the serum blocking agent is an antibody in the serum of an animal which is from the same source as the secondary antibody, and can be combined with a site which can be combined with the secondary antibody in a tissue in advance; or calf serum, BSA, sheep serum, and not consistent with the source of the primary antibody;
the buffer is PBS buffer;
the antibody diluent is PBS or a special antibody diluent added with a sodium azide preservative and/or a BSA stabilizer;
the dewaxing hydration agent and the dehydration clearing agent are xylene and ethanol; the ethanol comprises absolute ethanol, 95% ethanol, 85% ethanol and 75% ethanol.
7. A method of using the TIPE3 immunohistochemical detection kit of any one of claims 3-6 for immunohistochemical staining diagnosis of tissue sections, the method comprising:
s1, fixing the tissue to be detected of the person to be detected to prepare a paraffin slice;
s2, reacting the processed paraffin wax sheet with a primary antibody and a secondary antibody in sequence;
and S3, after the reaction is finished, cleaning the section, and observing the section through a microscope after DAB staining, hematoxylin counterstaining, dehydration and transparent sealing.
8. Use according to claim 7,
in step S1, the tissue to be measured of the subject is a pancreatic tissue;
in step S2, the processing procedure of the paraffin plate includes: dewaxing and hydrating the prepared paraffin wax tablets, performing antigen retrieval, inactivating peroxidase and biotin, and sealing serum;
in the step S3, the microscope observation includes: randomly selecting three fields for each sample, and each group comprises about 500 cells; semi-quantitative scoring (0, negative; 1, +; 2, + +; 3, + ++) based on hematoxylin staining intensity, on a four-point basis (< 1%, 0 min.; 1-25%, 1 min.; 26-50%, 2 min.; 60-75%, 3 min.; 76-100%, 4 min.) based on the percentage of positive cells; combining the two scores for each sample to obtain a final TIPE2 expression score; the cut-off points defining the expression levels are: 0-3 points, low expression; 4-8 points, high expression.
9. Use of the TIPE3 immunohistochemical detection kit and/or method of use in pancreatic cancer detection.
10. The use according to claim 9, wherein the tumor detection comprises in particular: judging the malignancy degree of the pancreatic cancer and the prognosis of the patient.
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