In-situ detection method of protein and phosphoinositide compound in tissue level

Abstract

The invention relates to the technical field of biological detection, and discloses an in-situ detection method for the tissue level of a protein and phosphoinositide compound, which comprises the following steps: providing a to-be-detected tissue sample containing the protein and phosphoinositide compound; incubating the sample to be detected by using a primary antibody mixture; incubating a product obtained in the previous step by using a second antibody mixture containing a PLA probe, and reacting to obtain circular DNA (Deoxyribose Nucleic Acid); carrying out in-situ amplification by taking the circular DNA as a template; and acquiring and analyzing a signal of the detection probe. According to the method, the interaction between the protein and the phosphoinositide compound can be specifically detected in situ at the tissue level, the actual state of the protein and the phosphoinositide compound in the physiological and pathological processes of cells can be more truly reflected by the technology, and more accurate information is provided for researching the function and regulation mechanism of the protein and the phosphoinositide compound; wide application prospects are realized.

Description

Technical Field

[0001] The present application relates to the field of biological detection technology, and in particular to a method for in situ detection of protein and phosphoinositide complexes at the tissue level. Background Art

[0002] Protein-phosphoinositide complexes play key roles in numerous cellular physiological processes, including cell signaling, gene expression regulation, and cell cycle control. For example, the interaction between the cancer suppressor protein p53 and the phosphoinositide lipid signaling pathway is crucial for maintaining cell life cycle and genomic stability, and its abnormalities can induce a variety of diseases, including cancer. However, current research on protein-phosphoinositide complexes is mostly limited to in vitro experiments, lacking effective methods for in situ detection of protein-phosphoinositide complexes at the cellular and tissue levels, greatly limiting our in-depth understanding of their mechanisms of action in cellular physiology and pathology.

[0003] In the prior art, immunohistochemistry (Immunohistochemistry, IHC) is an experimental method commonly used to detect the expression of specific proteins in tissue samples. It binds to the target protein through specific antibodies, and then uses staining technology to visualize the location and content of the protein in the tissue. Similarly, immunofluorescence (IF) is also mainly used for the detection of proteins. These two technologies are less used for the detection of small molecular lipids such as phosphoinositides, and cannot directly detect the interaction between proteins and phosphoinositides. Proximity ligation assay (PLA) is a bimolecular detection method based on complementary ligation of oligostranded DNA. It is widely used to study the interaction between proteins. It can highly specifically detect the physical proximity between target molecules (about 30-40nm apart). Its application range is mainly limited to the interaction between proteins. There is no mature solution for the detection of protein-lipid complexes. Therefore, the prior art needs to be improved. Summary of the Invention

[0004] In view of the above-mentioned deficiencies in the prior art, the purpose of this application is to provide a method for in situ detection of protein and phosphoinositide complexes at the tissue level, aiming to solve the problem in the prior art that it is impossible to perform in situ detection of protein and phosphoinositide complexes at the tissue level.

[0005] The technical solution of this application is as follows:

[0006] In a first aspect of the present application, a method for in situ detection of a protein-phosphoinositide complex at the tissue level is provided, comprising:

[0007] S1: providing a tissue sample to be tested, wherein the sample to be tested contains a protein and phosphoinositide complex;

[0008] S2: incubating the tissue sample to be tested with a primary antibody mixture, wherein the primary antibody mixture comprises two antibodies that specifically recognize proteins and phosphoinositides, respectively;

[0009] S3: Incubate the product in S2 with a secondary antibody mixture containing PLA probes. The secondary antibody mixture includes a ligase and two secondary antibodies with specific PLA probes. The two secondary antibodies can recognize the primary antibody in S2 and are respectively equipped with a PLA first nucleotide probe and a PLA second nucleotide probe. When the distance between the protein and phosphoinositide in the tissue sample to be tested is less than 40 nm, the PLA first nucleotide probe and the PLA second nucleotide probe will form circular DNA under the action of the ligase.

[0010] S4: performing roller amplification using the circular DNA as a template, wherein the amplification system includes a DNA polymerase; performing roller amplification using the circular DNA as a template under the action of the DNA polymerase;

[0011] S5: The detection probe is incubated with the product in S4 to obtain the detection probe signal, and the in situ detection result of the protein and phosphoinositide complex is obtained through analysis.

[0012] Optionally, the method for preparing the sample to be tested includes: providing a tissue to be tested, embedding the tissue to be tested in paraffin to obtain a tissue-embedded wax block, and slicing the tissue-embedded wax block to obtain the sample to be tested.

[0013] Optionally, the method for preparing the tissue sample to be tested further comprises the step of performing antigen repair treatment on the sample to be tested.

[0014] Optionally, the antigen repair treatment includes at least one of microwave heat treatment, water bath heat treatment, enzymatic repair heat treatment, and high-pressure heat repair treatment.

[0015] Optionally, when the antigen repair treatment is high-pressure heat repair treatment, the treatment temperature of the high-pressure heat repair treatment is 121° C. and the treatment time is 2 hours.

[0016] Optionally, one of the PLA first nucleotide probe and the PLA second nucleotide probe is a PLUS probe, and the other is a MINUS probe.

[0017] Optionally, the detection probe includes at least one of a fluorescent label, a radioactive label, and a chemiluminescent label.

[0018] Optionally, when the chemiluminescent label on the detection probe is horseradish peroxidase, the step of obtaining the detection signal of the detection probe comprises: adding a horseradish peroxidase (HRP) labeled nucleic acid probe to the product of the rolling circle amplification, then adding a horseradish peroxidase chromogenic substrate diaminobenzidine (DAB), and if a brown precipitate signal is detected, the protein-phosphoinositol complex is contained in the tissue sample to be tested.

[0019] Optionally, the protein is tumor protein p53, and the phosphoinositol is phosphatidylinositol triphosphate.

[0020] Compared with the prior art, the protein-phosphoinositol complex in situ detection method at the tissue level has the following advantages:

[0021] (1) The protein-phosphoinositol complex in situ detection method at the tissue level can specifically detect the interaction between the protein and the phosphoinositol, exclude the interference of random colocalization, significantly improve the specificity and sensitivity of the detection through a double recognition mechanism, and accurately detect the presence of the complex at a low expression level.

[0022] (2) The protein-phosphoinositol complex in situ detection method at the tissue level can detect the protein-phosphoinositol complex in situ at the tissue level, retains the integrity of the tissue and the intercellular relationship. Compared with the existing in vitro detection method, it can more truly reflect the actual state of the protein-phosphoinositol complex in the physiological and pathological processes of cells, and provide more accurate information for studying the function and regulation mechanism.

[0023] (3) The protein-phosphoinositol complex in situ detection method at the tissue level can restore the immunogenicity of the protein and the phosphoinositol at the same time by optimizing the antigen repair conditions, ensure the full exposure and combination of the two antigens in the tissue, overcome the limitations of the traditional antigen repair method in dealing with the complex, and improve the accuracy and stability of the detection results.

[0024] (4) The protein-phosphoinositol complex in situ detection method at the tissue level has relatively simple operation steps and is easy to implement in a conventional laboratory. Through a standardized experimental process, the repeatability and reliability of the experimental results can be ensured, and the experimental difficulty and cost are reduced.

[0025] (5) The in situ detection method of protein-phosphoinositide complexes at the tissue level provides a new approach for studying protein-lipid interactions, contributing to a deeper understanding of their mechanisms of action in cellular physiology and pathology. Furthermore, this method has broad application prospects, such as in the detection of tumor markers and drug screening, and will provide new strategies and methods for early diagnosis and personalized treatment of diseases. BRIEF DESCRIPTION OF THE DRAWINGS

[0026] In order to more clearly illustrate the technical solutions in the embodiments of the present application, the following briefly introduces the drawings required for describing the embodiments.

[0027] Figure 1 Schematic diagram of the principle of the in situ detection method of protein and phosphoinositide complex at the tissue level provided in the embodiments of the present application;

[0028] Figure 2 A schematic diagram of the process of the in situ detection method of protein and phosphoinositide complex at the tissue level provided in the embodiments of the present application;

[0029] Figure 3 Observation diagram of the human tumor cell line xenogeneic mouse model and tumor tissue provided in the examples of this application;

[0030] Figure 4 This is an in situ microscopic observation image (20x objective lens) of the tumor protein p53 and phosphatidylinositol triphosphate (PtdIns(3,4,5)P3) / phosphatidylinositol bisphosphate (PtdIns(4,5)P2) complex provided in the examples of the present application at the tissue level. DETAILED DESCRIPTION

[0031] To make the purpose, technical solutions and advantages of this application clearer, the technical solutions in the embodiments of this application will be clearly and completely described below in conjunction with the drawings and embodiments of this application. Obviously, the described embodiments are only part of the embodiments of this application, not all of the embodiments. Based on the embodiments of this application, all other embodiments obtained by ordinary technicians in this field without making creative work are within the scope of protection of this application. In the absence of conflict, the features in the following embodiments and embodiments can be combined with each other.

[0032] It should be noted that if there are descriptions involving "first", "second", etc. in the implementation of this application, the descriptions such as "first", "second", etc. are only used for descriptive purposes and cannot be understood as indicating or suggesting their relative importance or implicitly indicating the number of the indicated technical features. Therefore, the features defined as "first" and "second" may explicitly or implicitly include at least one of such features. In addition, the technical solutions between the various embodiments can be combined with each other, but they must be based on enabling ordinary technicians in this field to implement them. When the combination of technical solutions is contradictory or cannot be implemented, it should be deemed that such a combination of technical solutions does not exist and is not within the scope of protection required by this application.

[0033] The existing technologies for detecting protein-phosphoinositide complexes have the following problems and shortcomings:

[0034] First, the lack of effective experimental methods for in situ detection of protein-phosphoinositide complexes at the tissue level limits the study of their mechanisms of action in cellular physiological and pathological processes. Second, existing immunofluorescence and proximity ligation techniques have limitations in detecting protein-phosphoinositide complexes and cannot meet the detection needs of such complexes. In addition, traditional antigen retrieval methods have limitations in processing protein-phosphoinositide complexes. In immunotissue staining, antigen retrieval is one of the key steps used to restore the immune activity of masked or inactivated antigens. However, existing antigen retrieval methods mainly target a single type of antigen, and their retrieval effect is not ideal for complexes containing both proteins and lipids, resulting in difficulty in obtaining accurate and stable results when detecting protein-phosphoinositide complexes at the tissue level.

[0035] The existence of the above problems and shortcomings is mainly due to the complex dynamic distribution and interaction of proteins and phosphoinositide complexes in cells, and current technical means are difficult to simultaneously meet the specific detection of proteins and lipids and the accurate identification of the interactions between them.

[0036] Based on this, see Figure 1 and Figure 2 The present invention provides an in situ detection method of protein and phosphoinositide complex at the tissue level, comprising the steps of:

[0037] S1. Provide a tissue sample containing a protein and phosphoinositide complex.

[0038] In some embodiments, the method for preparing a sample to be tested of a protein and phosphoinositide complex comprises: providing a tissue to be tested, and preparing the tissue to be tested into paraffin sections.

[0039] Specifically, after obtaining the tissue to be tested, the tissue to be tested can be embedded in paraffin to obtain a tissue-embedded wax block, and then the tissue-embedded wax block can be sliced ​​to obtain paraffin slices.

[0040] In some embodiments, the method for preparing a tissue sample to be tested for protein and phosphoinositide complexes further comprises: performing antigen retrieval on the sample to be tested obtained in S1. Furthermore, the antigen retrieval comprises at least one of microwave heat treatment, water bath heat treatment, enzymatic repair heat treatment, and high-pressure heat repair treatment.

[0041] Traditional antigen repair treatment is only used for protein processing, and its detection effect on protein and phosphoinositide complexes remains to be explored. The examples of this application compare microwave heat repair, water bath heat repair, enzymatic repair and high-pressure heat repair. Preferably, high-pressure heat repair treatment can best expose the two types of antigens.

[0042] In some embodiments, the 2100 Antigen Retriever device is used for antigen high-pressure heat repair treatment, the treatment temperature is 121° C., and the entire treatment cycle is 2 hours.

[0043] S2. Incubate the sample with a primary antibody cocktail containing two antibodies that specifically recognize different antigen types (protein and phosphoinositide). Incubate the tissue sample with a protein-phosphoinositide complex using a primary antibody cocktail containing two antibodies from different species that specifically recognize the specific protein and phosphoinositide in the complex. Both antibodies are commercially available for IF and IHC, and there is no cross-reactivity between the two, so screening for antibody specificity is required.

[0044] S3. Incubate the product in S2 with a PLA secondary antibody mixture. The secondary antibody mixture includes a ligase and two secondary antibodies with specific PLA probes. The two secondary antibodies can recognize the primary antibody in S2, and the two secondary antibodies are respectively equipped with a PLA first nucleotide probe and a PLA second nucleotide probe. When the distance between the protein and the phosphoinositide in the tissue sample to be tested is less than 40 nm, the PLA first nucleotide probe and the PLA second nucleotide probe will form circular DNA under the action of the ligase.

[0045] In some embodiments, one of the PLA first probe antibody and the PLA second probe antibody is a PLUS probe, and the other is a MINUS probe. The PLUS probe and the MINUS probe are usually composed of two parts: one part is an antibody or an aptamer capable of specifically recognizing different species; the other part is an oligonucleotide chain connected to the antibody or the aptamer. The two oligonucleotide chains have different sequence characteristics, corresponding to the identification of PLUS and MINUS, respectively. When the secondary antibodies containing the PLUS probe and the MINUS probe recognize the first antibody connected to the protein and the phosphoinositide, respectively, and the PLUS probe and the MINUS probe are close to a certain extent (less than 40 nm), the probes on the two antibodies are connected into a complete circular DNA molecule under the action of a ligase.

[0046] S4, amplifying at the target complex with the circular DNA as a template, wherein the amplification system comprises a DNA polymerase, and the DNA polymerase performs rolling circle amplification with the circular DNA as a template.

[0047] S5, detecting the circular DNA, and obtaining an in-situ detection result of the protein-phosphoinositide complex through analysis.

[0048] The detection probe carries a detection label, and a part of the nucleotide sequence of the detection probe is complementary to a part of the nucleotide sequence of the amplified DNA.

[0049] In some embodiments, the detection label comprises at least one of a fluorescent label, a radioactive label, and a chemiluminescent label. The fluorescent label includes, but is not limited to, FAM, TAMRA, Cy3, Cy5, etc. The radioactive label includes, but is not limited to 32 P, 3 H, 14 C, etc. The chemiluminescent label includes, but is not limited to, horseradish peroxidase, luminol and its derivatives, acrid esters, etc. The detection label can also be biotin, digoxin, etc.

[0050] Specifically, while the circular DNA is amplified, the detection probe can hybridize with the complementary sequence on the amplified DNA sequence. For example, when the detection probe is fluorescently labeled, the presence and quantity of the protein-phosphoinositide complex and the interaction between the protein and the phosphoinositide can be determined by detecting the fluorescent signal. For another example, when the detection probe includes a chemiluminescent marker, and the chemiluminescent marker is horseradish peroxidase (HRP), the step of obtaining the detection signal of the detection probe includes: adding a nucleic acid probe labeled with HRP to the product of the roller amplification, and then adding an HRP color development substrate (DAB), the substrate DAB reacts with H2O2, and an insoluble brown product is generated by transferring electrons from DAB to peroxide. The brown precipitate signal is detected, indicating that the tissue sample to be tested contains a protein-phosphoinositide complex.

[0051] The principle of the in situ detection method of protein and phosphoinositide complexes at the tissue level in the examples of the present application is as follows: tissue sections are incubated simultaneously with primary antibodies that specifically recognize proteins and phosphoinositides; when the target protein interacts with the phosphoinositide to form a complex, a detectable DNA linker is formed by the action of a ligase on the PLA first nucleic acid probe and the PLA second nucleic acid probe attached to the two PLA probe-containing secondary antibodies; the DNA sequence on the linker is amplified by a PCR reaction, and detection is performed using an oligostranded probe (detection probe) complementary to the amplified DNA sequence, thereby visualizing the presence and distribution of the protein and phosphoinositide complex in situ in the tissue.

[0052] Although current IHC technology can detect protein expression in tissue, it lacks the detection method for small molecule lipids such as phosphoinositides, so it is impossible to directly detect the interaction between protein and phosphoinositides in situ. Although PLA technology can detect the interaction between proteins, there is no mature method to be applied to the detection of protein and lipid complex. The protein and phosphoinositide complex in the present application embodiment combines the advantages of IHC and PLA in the in situ detection method of tissue level, has both achieved the labeling of protein and phosphoinositide at the same time, and has utilized the principle of PLA technology to detect the interaction between protein and phosphoinositide. Significantly improve the specificity and sensitivity of detection by this dual recognition mechanism, so that even at low expression levels, the presence of complex can be accurately detected, and the interaction of protein and phosphoinositide in complex can be detected.

[0053] In some embodiments, the protein is the tumor protein p53 and the phosphoinositide is phosphatidylinositol triphosphate.

[0054] The in situ detection method for protein-phosphoinositide complexes at the tissue level described in the examples of this application can be used for research in areas such as cell signaling, gene expression regulation, and cell cycle control. This complex plays an important role in these physiological processes, and this detection method can accurately detect their interactions, facilitating research into related mechanisms.

[0055] The in situ detection method for protein-phosphoinositide complexes at the tissue level provided in the examples of this application provides a new approach for studying protein-lipid interactions, contributing to a deeper understanding of their mechanisms of action in cellular physiology and pathology. Furthermore, this method has great potential for clinical applications, such as tumor marker detection and drug screening, and will provide new strategies and methods for early diagnosis and personalized treatment of diseases.

[0056] The following is further described with reference to specific examples.

[0057] Example 1 Construction of a human tumor cell line xenograft mouse model

[0058] To detect protein-phosphoinositide interactions at the tissue level in situ, a human breast cancer cell line (MDA-MB-231) was used to construct a tumor-bearing mouse model. The MDA-MB-231 cell line was provided by the Cell Bank of the Chinese Academy of Sciences. The cell line was cultured in DMEM medium (containing 10% fetal bovine serum and 1% streptomycin / penicillin) and subcultured in a 37°C, 5% CO2 incubator. Five-week-old immunodeficient Balb / c nude mice were used. To obtain statistically significant experimental results, three female mice were required. The specific steps included:

[0059] Adjust the cell density to 5 × 10 7 100 μL of MDA-MB-231 cells were inoculated subcutaneously on both sides of the axilla of female nude mice. When the tumor volume reached 80-100 mm 3 When (such as Figure 1 The mice were anesthetized with isoflurane and samples were taken. The tumor tissues were fixed with 4% paraformaldehyde fixative (Biyuntian, P0099) for 12 hours and then used to prepare tissue sections.

[0060] Example 2 Slice Preparation

[0061] (1) Tissue block embedding

[0062] The tumor tissue obtained in Example 1 was dehydrated and waxed using an automatic biological tissue dehydrator (KD, KD-TS3A). The specific parameters were set as follows:

[0063]

[0064] Subsequently, the tissue blocks were embedded in paraffin using a paraffin embedding machine (Kodi, KD-BM / BL).

[0065] (2) Slicing

[0066] Tumor tissue paraffin blocks were sectioned to a thickness of 5 μm using a paraffin slicer (Rayward, S700A). The wax strips were spread with the glossy side facing down on the surface of the slide spreader. The sections were then scooped up with a glass slide and placed in the center of the slide. The slides with the wax strips were immediately dried in a 60°C incubator for 2 hours to initially melt away excess paraffin on the sections.

[0067] Then, dewax the slices in xylene tanks I and II for 20 minutes (shaking can accelerate dewaxing, and warm dewaxing is more thorough; if the room temperature is low, the dewaxing time can be appropriately extended). Then, rehydrate with 100%, 100%, 95%, 80%, 70%, and 50% ethanol, soaking for 5 minutes each step, and then rinse three times with deionized water.

[0068] Example 3 Antigen retrieval

[0069] Antigen repair can fully open the antigen protein cross-linked by formaldehyde in the fixative to restore the antigenicity of the protein. The specific steps are: take the slices prepared in Example 2, wash them again 3 times with PBS, and then use 2100AntigenRetriever (Aptum, Pick Cell Laboratories) for antigen repair. First place the slices on a glass slide and place them in the equipment slide box, pour in about 70-100mL of sodium citrate antigen repair buffer (Biyuntian, P0081), the processing temperature is 121°C, and the entire processing cycle is 2 hours. Take out the glass slides that have been naturally cooled to room temperature from the repair buffer, soak the slides twice with deionized water, and then soak them again with TBS buffer 3 times.

[0070] Example 4 In situ detection of protein and phosphoinositide complexes at the tissue level

[0071] Using the interaction between phosphatidylinositol triphosphate (PtdIns(3,4,5)P3) and the tumor protein p53 as an example, a method for detecting the interaction between phosphoinositides and proteins was established. Phosphoinositides consist of an inositol ring and two fatty acid chains connected by a glycerol backbone. Phosphoinositides are formed into various isoforms through phosphorylation at the 3rd, 4th, or 5th hydroxyl groups of the inositol ring. Therefore, screening for phosphoinositide antibodies with strong specificity and suitability for tissue staining will be crucial for the development of this technology. Currently available PtdIns(3,4,5)P3 antibodies include Anti-PtdIns(3,4,5)P3 IgM (Echelon Biosciences, Z-P345) and Anti-PtdIns(3,4,5)P3 IgG (Echelon Biosciences, Z-P345B). Anti-PtdIns(3,4,5)P3IgM is more suitable for in situ PtdIns(3,4,5)P3 detection, while Anti-PtdIns(3,4,5)P3 IgG is more suitable for ELISA and other experiments. The experimental procedure is as follows:

[0072] (1) After the slices were rewarmed, PBS containing 0.3% Triton X-100 was added dropwise to permeabilize the tissue for 30 minutes, and then TBST buffer containing 0.1% Tween-20 was added to wash the slices three times, each time for 5 minutes.

[0073] (2) Add an appropriate amount of 3% normal goat serum (diluted with TBS) and block at room temperature for 1 hour.

[0074] (3) Discard the blocking solution and add diluted mixed primary antibodies of different origins, namely Anti-PtdIns (3,4,5) P3IgM (Echelon Biosciences, Z-P345) and Anti-p53 IgG (Cell Signaling Technology, 2527S). The antibodies were diluted with universal antibody diluent (AboRo, RK0202) and incubated in a humidified chamber at 4°C overnight.

[0075] (4) Binding of secondary antibodies containing PLA probes to primary antibodies: Two secondary antibodies containing PLA probes of different origins, namely PLUSprobe antibody and MINUS probe antibody, were diluted with Ab diluent (1:5). The incubation volume was adjusted according to the size of the tissue. The slides were placed in a humidified chamber and incubated at 37°C for 1 hour. Unbound PLA probes were washed away with TBST buffer containing 0.1% Tween-20, and the washing was performed three times for 5 minutes each time.

[0076] (5) PLA probe ligation reaction: Dilute 5× Ligation (ligation reaction buffer) to 1× with ddH2O to obtain dilution solution I. Then, dilute T4 DNA Ligase to a concentration of 20 units / μL with dilution solution I to obtain reaction solution I. Add reaction solution I dropwise onto a glass slide, place the slide in a humidified chamber, and incubate at 37°C for 30 minutes. Wash away the reaction solution with TBST buffer containing 0.1% Tween-20, and rinse three times for 5 minutes each time.

[0077] (6) Probe Amplification: Dilute 5× Amplification Red to 1× with ddH2O to obtain diluent II. Then, dilute Phi29 DNA Polymerase to a concentration of 0.5 units / μL with diluent II to obtain reaction solution II. Add reaction solution II dropwise onto a glass slide, place the slide in a humidified chamber, and incubate at 37°C for 100 minutes.

[0078] (7) Use TBS to wash away the reaction solution, add freshly prepared DAB color development solution on the slice, observe under a microscope, control the color development time, and after the color development is completed, use PBS to wash away the color development solution to stop the color development.

[0079] (8) After color development, re-stain with hematoxylin for 5 minutes, rinse with running water, then place in hydrochloric acid alcohol for a few seconds and then rinse with running water again, and finally place in ammonia water to return to blue.

[0080] (9) Seal the slides with neutral gum and observe and record under a microscope.

[0081] The experimental results are as follows Figure 2 The results show that the complex of tumor protein p53 and phosphatidylinositol triphosphate (PtdIns(3,4,5)P3) (brown) can be observed in the sections under a microscope, indicating that this technology has achieved in situ detection of the complex of tumor protein p53 and phosphatidylinositol triphosphate (PtdIns(3,4,5)P3) at the tissue level. At the same time, we also provide the results of in situ detection of the complex of tumor protein p53 and phosphatidylinositol bisphosphate (PtdIns(4,5)P2) at the tissue level.

[0082] In summary, the in situ detection method of proteins and phosphoinositide complexes at the tissue level in the examples of this application, combined with the advantages of immunofluorescence and proximity ligation techniques, overcomes the limitations of existing technologies in detecting proteins and phosphoinositide complexes. By optimizing antigen retrieval conditions and screening specific antibodies, this method can detect proteins and phosphoinositide complexes in situ at the tissue level, providing a powerful tool for studying the mechanism of protein-lipid interaction and has broad application prospects.

[0083] It should be understood that the application is not limited to the examples described above, which can be modified or transformed by a person of ordinary skill in the art in light of the above description, all these modifications and transformations being intended to belong to the scope of protection of the claims appended to the application.

Claims

1. A method for in situ detection of protein and phosphoinositide complexes at the tissue level, characterized in that: Including steps: S1: providing a tissue sample to be tested, wherein the tissue sample to be tested contains a protein and phosphoinositide complex; S2: incubating the tissue sample to be tested with a primary antibody mixture, wherein the primary antibody mixture comprises two antibodies that specifically recognize proteins and phosphoinositides, respectively; S3: Incubating the product in S2 with a secondary antibody mixture containing PLA probes, wherein the secondary antibody mixture includes a ligase and two secondary antibodies with specific PLA probes, wherein the two secondary antibodies can respectively recognize the primary antibody in S2 and carry a PLA first nucleotide probe and a PLA second nucleotide probe, respectively; when the distance between the protein and the phosphoinositide in the tissue sample to be tested is less than 40 nm, the PLA first nucleotide probe and the PLA second nucleotide probe will form circular DNA under the action of the ligase; S4: performing roller amplification using the circular DNA as a template, wherein the amplification system includes a DNA polymerase; performing roller amplification using the circular DNA as a template under the action of the DNA polymerase; S5: The detection probe is incubated with the product in S4 to obtain the detection probe signal, and the in situ detection result of the protein and phosphoinositide complex is obtained through analysis.

2. The method for in situ detection of protein and phosphoinositide complexes at the tissue level according to claim 1, characterized in that: The method for preparing the tissue sample to be tested comprises: providing the tissue to be tested, embedding the tissue to be tested in paraffin to obtain a tissue-embedded wax block, and slicing the tissue-embedded wax block to obtain the tissue sample to be tested.

3. The method for in situ detection of protein and phosphoinositide complexes at the tissue level according to claim 1, characterized in that: The method for preparing the tissue sample to be tested further comprises the step of performing antigen repair treatment on the sample to be tested.

4. The method for in situ detection of protein and phosphoinositide complexes at the tissue level according to claim 3, characterized in that: The antigen repair treatment includes at least one of microwave heat treatment, water bath heat treatment, enzymatic repair heat treatment, and high-pressure heat repair treatment.

5. The method for in situ detection of protein and phosphoinositide complexes at the tissue level according to claim 4, characterized in that: The treatment temperature of the high pressure thermal repair treatment is 121° C. and the treatment time is 2 hours.

6. The method for in situ detection of protein and phosphoinositide complexes at the tissue level according to claim 1, characterized in that: One of the PLA first nucleotide probe and the PLA second nucleotide probe is a PLUS probe, and the other is a MINUS probe.

7. The method for in situ detection of protein and phosphoinositide complexes at the tissue level according to claim 1, characterized in that: The detection probe includes at least one of a fluorescent label, a radioactive label, and a chemiluminescent label.

8. The method for in situ detection of protein and phosphoinositide complexes at the tissue level according to claim 7, characterized in that: When the detection probe includes a chemiluminescent label, and the chemiluminescent label is horseradish peroxidase, the step of obtaining the detection signal of the detection probe includes: A nucleic acid probe labeled with horseradish peroxidase is added to the roller amplification product, and then a horseradish peroxidase color development substrate is added. If a brown precipitation signal is detected, the tissue sample to be tested contains a protein and phosphoinositide complex.

9. The method for in situ detection of protein and phosphoinositide complexes at the tissue level according to claim 1, characterized in that: The protein is tumor protein p53, and the phosphoinositide is phosphatidylinositol triphosphate.

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