A kit for detecting ligands of nkg2d receptor and cd56 and use thereof

Abstract

The application discloses a kit for detecting ligands of NKG2D receptors and CD56 and application thereof. The kit comprises the following components: an antibody eluent, a primary antibody group and a multiplex fluorescence immunohistochemical reagent group. The application is based on tyramide signal amplification (TSA) technology, realizes labeling of biomarkers of a plurality of ligand families of NKG2D receptors and NK cell markers CD56 in tumor tissues on the same tissue section, and uses a low-temperature antibody eluent to elute the antibodies in the staining process, thereby avoiding the problem of slice peeling caused by multiple antigen repairs. The application also provides a use method of the kit on an automatic staining instrument. The method is simple to use, low in labor cost and high in repeatability, and can meet the requirements of scientific research and clinical detection in high precision and high accuracy.

Description

Technical Field

[0001] This invention belongs to the field of biodetection technology, specifically relating to a kit for detecting ligands and CD56 of the NKG2D receptor and its application. Background Technology

[0002] Immunohistochemistry (IHC) is a technique that utilizes the principle of specific binding between antigens and antibodies to identify intracellular antigens through chemical reactions that bring labeling agents to color or through fluorescein staining, thereby enabling the localization, qualitative analysis, and quantification of these antigens. Due to its advantages such as high specificity, high sensitivity, accurate localization, integration of morphology and function, and simplicity and speed, it has been widely used in life sciences, translational medicine research, and clinical diagnosis, particularly in tumor diagnosis, for many years. With the continuous development of tumor biology research and the increasing research into targeted tumor drugs, the demand for the detection of tumor immune-related biomarkers is also constantly increasing, and IHC, which can only detect single biomarkers, can no longer meet this need. Building upon traditional IHC, this technique combines tyramine signal amplification (TSA) technology with cyclic staining to achieve co-staining of multiple biomarkers on the same tissue section. This not only satisfies the traditional IHC requirements for the localization, qualitative analysis, and quantification of single biomarkers but also allows for the study of their co-localization, expression levels, and interactions.

[0003] Natural killer (NK) cells represent the forefront of immunotherapy strategies, and various therapeutic approaches that fully utilize the anti-tumor potential of NK cells are being gradually applied. One of the NK cell activation receptors closely related to NK cell therapy is NKG2D, which recognizes eight different NKG2D ligands (NKG2DL), including members of the MIC family (MICA, MICB) and the ULBP family (ULBP1–ULBP6). Currently, NKG2D ligands (NKG2DL) are being used as molecular targets in immuno-oncology. Developing multiplex detection technologies targeting NKG2D ligands, capable of simultaneously quantifying the expression levels of multiple ligands, has significant application value in the field of tumor treatment and can serve as an indicator for predicting and evaluating the efficacy of immunotherapy. NKG2D ligand expression levels are a key indicator of tumor cell immunogenicity. Multiplex detection can simultaneously assess the expression profiles of multiple ligands, accurately predicting the response rate of NK cell or CAR-NK therapy.

[0004] Existing methods for detecting tumors using TSA fluorescent dyes in multiplex immunohistochemical assays (IHCs) include Chinese patent application CN116008549A, filed on December 29, 2022, entitled "A Multiplex Fluorescent Immunohistochemical Detection Kit and Its Application." This method uses branched TSA dyes instead of DAB dyes in traditional IHC, and through multiple rounds of staining, it can fluorescently label up to eight different antigens on the same tissue section. Simultaneously, it combines a full-spectrum fluorescence imaging system and spectral splitting imaging software to image tissue sections with up to nine colors (including cell nuclei). However, this approach uses microwave antigen retrieval methods that are repeatedly applied, which may cause tissue section detachment and thermal loss of antigenic epitopes, and it does not consider the correspondence between antigenic epitope abundance and fluorescent dye signals. For example, Chinese patent application publication number CN111830256A, entitled "A Paraffin Section Immunofluorescence Multiplex Staining Kit and Method of Use," employs TSA signal amplification technology. After the primary antibody binds to the antigen, it is incubated with an HRP-labeled secondary antibody. HRP catalyzes the addition of a fluorescein substrate to the system, generating an activated fluorescent substrate. The activated substrate can covalently bind to tyrosine residues on the antigen, resulting in stable covalent binding of fluorescein to the sample. Then, heat retrieval washes away the non-covalently bound antibody, and the cycle is repeated to achieve multiplex labeling. However, this method requires stepwise staining, first catalyzing the biotin-Tyramide linkage, then using Strepavidin dye. The operation is complex, requires high manual labor, and does not consider the correspondence between antigen epitope abundance and fluorescent dye signal. Summary of the Invention

[0005] The technical problem this invention aims to solve is that existing multiplex fluorescence immunohistochemistry methods lack specific methods for NKG2D receptor ligands and CD56-related biomarkers. Furthermore, most cyclic staining methods based on tyramine signal amplification (TSA) technology use microwave or high-temperature antigen retrieval to elute antibodies, neglecting potential issues such as slide detachment and antigen loss from repeated high-temperature treatments. This application provides a kit for evaluating NKG2D receptor ligands and CD56 and its application. Based on tyramine signal amplification (TSA) technology, multiple NKG2D receptor ligands and CD56 biomarkers in tumor tissue can be labeled on the same tissue section. The kit can be used on an automated staining instrument, and by combining it with a full-spectrum fluorescence imaging system, whole-slide imaging analysis can be performed. This method is simple to use, has low labor costs, and high reproducibility, meeting the high precision and accuracy requirements of scientific research and clinical testing.

[0006] The present invention solves the above-mentioned technical problems through the following technical solutions.

[0007] The first aspect of the present invention provides a kit for detecting ligands and CD56 of the NKG2D receptor, the kit comprising the following components: antibody eluent, primary antibody group and multiplex fluorescent immunohistochemistry reagent group; the primary antibody group comprises specific antibodies for six biomarkers: ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4, MIC A / B and CD56;

[0008] The multiplex fluorescent immunohistochemical reagent set includes a set of TSA fluorescent dyes that correspond one-to-one with the specific antibodies.

[0009] The antibody elution buffer is used for elution after staining with the specific antibody.

[0010] In some embodiments, the TSA fluorescent dye group includes: TSA480, TSA520, TSA570, TSA620, TSA700, TSA770 and DAPI.

[0011] In some implementations, the TSA520 corresponds to the ULBP4 antibody; the TSA700 corresponds to the ULBP3 antibody; the TSA620 corresponds to the ULBP2 / 5 / 6 antibody; the TSA480 corresponds to the MIC A / B antibody; the TSA570 corresponds to the ULBP1 antibody; and the TSA770 corresponds to the CD56 antibody.

[0012] In some specific implementations, the ULBP3 antibody, MIC A / B antibody, ULBP1 antibody, and CD56 antibody were purchased from Abcam, with catalog numbers ab89931, ab224702, ab238331, and ab200698, respectively; the ULBP4 antibody and ULBP2 / 5 / 6 antibody were purchased from R&D systems, with catalog numbers MAB6285 and MAB1298, respectively.

[0013] In some embodiments, the multiplex fluorescent immunohistochemistry reagent kit further includes a signal amplification reaction solution, a mouse-rabbit mixed HRP-labeled secondary antibody, and a blocking solution.

[0014] In some embodiments, the antibody eluent is an antibody eluent with an operating temperature of 37-50°C, such as Absin antibody eluent with catalog number Abs994.

[0015] In some implementations, the washing solution is a TBS washing solution.

[0016] In some embodiments, the kit further includes a dewaxing solution, an antigen retrieval solution, a washing solution, a 0.3%-5% hydrogen peroxide solution (e.g., a 3% hydrogen peroxide solution), an ethanol solution, and an anti-fluorescence quenching mounting medium.

[0017] In some implementations, the antigen retrieval solution is an alkaline antigen retrieval solution with a pH value of 8.9-9.1.

[0018] In some embodiments, the alkaline antigen retrieval solution is an EDTA solution.

[0019] A second aspect of the present invention provides a method for detecting ligands and CD56 of the NKG2D receptor, wherein the method uses a kit as described in the first aspect of the present invention to stain FFPE sections after incubation with primary and secondary antibodies.

[0020] In some embodiments, the primary antibody incubation is performed sequentially in the order of anti-ULBP4, ULBP3, ULBP2 / 5 / 6, MIC A / B, ULBP1, and CD56 specific antibodies.

[0021] In some embodiments, the secondary antibody used for incubation is a hybrid HRP-labeled secondary antibody (such as a mouse-rabbit antibody).

[0022] In some embodiments, the FFPE sections are rinsed with a washing solution after incubation with the primary antibody and the secondary antibody, but before staining.

[0023] In some embodiments, the FFPE slices are FFPE slices that have undergone a sealing treatment; the sealing treatment involves incubating the FFPE slices with a 0.3-5% hydrogen peroxide solution, such as a 3% hydrogen peroxide solution, and then incubating them in a sealing solution at room temperature.

[0024] In some embodiments, the blocked FFPR slices undergo antigen retrieval treatment before incubation in a 0.3-5% hydrogen peroxide solution, such as a 3% hydrogen peroxide solution.

[0025] In some embodiments, the antigen retrieval treatment involves placing the FFPE slide in an antigen retrieval solution and retrieval at 98°C followed by rinsing.

[0026] In some embodiments, the FFPE slides used for the antigen retrieval treatment are pretreated slides, the pretreatment being drying and dewaxing.

[0027] In some embodiments, the FFPE sections used for the antigen retrieval treatment are dewaxed FFPE sections.

[0028] In some implementations, the method includes the following steps:

[0029] Step 1: Place the slices in a 60℃ constant temperature oven and bake for 3 hours to obtain dried slices;

[0030] Step 2: Place the dried sections in a dewaxing solution at 72°C for 1 minute, wash 3 times with ethanol solution, and wash 3 times with washing solution to obtain pretreated sections;

[0031] Step 3: Place the pretreated slides in antigen retrieval solution at 98℃ for 20 minutes, and wash them 5 times with washing solution to obtain the retrieval slides;

[0032] Step 4: Incubate the repaired slices in a 3% hydrogen peroxide solution at room temperature for 15 minutes, and wash them 3 times with washing solution to obtain hydrogen peroxide-treated slices;

[0033] Step 5: Place the hydrogen peroxide-treated sections in a blocking solution at room temperature and incubate for 15 minutes to obtain the blocked sections;

[0034] Step 6: Incubate the blocked sections with primary antibody solution at 37°C for 1 hour, and wash with washing buffer 4 times to obtain sections incubated with primary antibody.

[0035] Step 7: After incubation with primary antibody, the slides are incubated with HRP-labeled secondary antibody (a hybrid of mouse and rabbit antibodies) at room temperature for 15-30 minutes, and then washed 4 times with washing buffer to obtain slides incubated with secondary antibody.

[0036] Step 8: After incubation with secondary antibody, the sections are incubated with TSA fluorescent dye at room temperature for 15-30 minutes, and then washed 4 times with washing solution to obtain stained sections;

[0037] Step 9: Incubate the stained sections in preheated antibody elution buffer at 37°C for 20 minutes, and wash 3 times with the washing buffer.

[0038] Step 10: Repeat steps 5-9 four times, then repeat steps 5-8 once to label the six biomarkers and obtain six stained sections.

[0039] Step 11: After 6 stainings, incubate the sections with DAPI at room temperature for 5-20 minutes, wash 3 times with washing buffer, and wash 3 times with sterile water to obtain the stained sections.

[0040] Step 12: Mount the stained sections using an anti-fluorescence quenching mounting medium to obtain the mounted sections;

[0041] Step 13: Use a full-spectrum fluorescence imaging system to photograph the slides after mounting, and obtain images of the fluorescence channels corresponding to each biomarker in the whole tissue slide and the merged fluorescence images.

[0042] In some implementations, the primary antibody is incubated in the order of ULBP4, ULBP3, ULBP2 / 5 / 6, MIC A / B, ULBP1 and CD56 as described in step 6.

[0043] In some specific embodiments, the TSA fluorescent dyes are incubated with the TSA fluorescent dyes described in step 8 in the order of TSA520, TSA700, TSA620, TSA480, TSA570 and TSA770.

[0044] In some specific implementations, the correspondence between the primary antibody and the fluorescent dye is as follows: TSA520 corresponds to ULBP4 antibody; TSA700 corresponds to ULBP3 antibody; TSA620 corresponds to ULBP2 / 5 / 6 antibody; TSA480 corresponds to MIC A / B antibody; TSA570 corresponds to ULBP1 antibody; and TSA770 corresponds to CD56 antibody.

[0045] In some implementations, imaging is performed using the Akoya Vectra Polaris full-spectrum fluorescence imaging system, which can obtain full-image fluorescence images of six biomarkers.

[0046] A third aspect of the present invention provides the application of a reagent kit as described in the first aspect of the present invention in the detection of human breast cancer.

[0047] In some implementations, the detection is for non-diagnostic purposes, such as assessing the content of NKG2D receptor ligands and CD56 in FFPE slices to determine whether the FFPE slices meet preparation or detection standards for drug screening.

[0048] Based on common knowledge in the field, the above-mentioned preferred conditions can be combined arbitrarily to obtain various preferred embodiments of the present invention.

[0049] The reagents and raw materials used in this invention are all commercially available.

[0050] The positive and progressive effects of this invention are as follows:

[0051] This invention provides a kit for evaluating NKG2D receptor ligands and CD56. While taking into account the high specificity, high sensitivity and accurate localization of traditional IHC technology, (1) based on tyramine signal amplification (TSA) technology, it is possible to label multiple NKG2D receptor ligands and CD56 biomarkers in tumor tissue on the same tissue slice. This avoids sample waste and can also analyze the co-localization expression, spatial location and grouping of biomarkers; (2) during the staining process, a low temperature antibody elution buffer is used instead of the microwave repair process to elute the antibody, avoiding the problem of slice detachment that may be caused by multiple microwave antigen repairs.

[0052] This invention provides a method for using a kit to evaluate NKG2D receptor ligands and CD56 on automated staining instruments. Based on the expression characteristics of six biomarkers and the performance differences of different fluorescent dyes, the optimal matching and incubation sequence of antibodies and fluorescent dyes were designed. By combining this with a full-spectrum fluorescence imaging system, the analysis of whole tissue sections for NKG2D ligands and six CD56-related biomarkers was completed. This method is simple to use, low in labor costs, and highly reproducible, fully meeting the high precision and accuracy requirements of scientific research and clinical testing. Attached Figure Description

[0053] Figure 1 The image shows the fluorescence of the whole tissue region where the six biomarkers provided in the example are co-expressed.

[0054] Figure 2 The fluorescence image of the biomarker MIC A / B provided in the example is shown in the 480nm fluorescence channel.

[0055] Figure 3 The fluorescence image of the biomarker ULBP4 provided in the example is shown in the 520nm fluorescence channel.

[0056] Figure 4 The fluorescence image of the biomarker ULBP1 provided in the example is shown in the 570nm fluorescence channel.

[0057] Figure 5 The fluorescence image of the biomarkers ULBP2 / 5 / 6 provided in the examples is shown in the 620nm fluorescence channel.

[0058] Figure 6 The fluorescence image of the biomarker ULBP3 provided in the example is shown in the 700nm fluorescence channel.

[0059] Figure 7 The fluorescence image of the biomarker CD56 provided in the example is shown in the 770nm fluorescence channel. Detailed Implementation

[0060] The present invention is further illustrated below by way of embodiments, but the invention is not limited to the scope of the embodiments described herein. Experimental methods in the following embodiments that do not specify specific conditions were performed according to conventional methods and conditions, or as selected according to the product instructions.

[0061] Example 1

[0062] This embodiment describes the use of a kit for evaluating ligands and CD56 of the NKG2D receptor, which can be used on an automated staining instrument. The slides used are human breast cancer FFPE samples (Sinotec Biotechnology). The specific operation of this embodiment is as follows.

[0063] The reagents used in the examples are shown in Table 1.

[0064] Table 1 Reagent Information

[0065]

[0066]

[0067] The instruments and equipment used in the embodiments are shown in Table 2.

[0068] Table 2. Instrument and Equipment Information

[0069]

[0070] The operation steps in the example are as follows:

[0071] 1. Baking the sheets:

[0072] Insert the slides into the slide holder and place them in a 60°C oven for 1 hour ± 10 minutes. Remove them and let them cool to room temperature.

[0073] 2. Solution preparation:

[0074] The primary antibody solution was prepared. The fluorescent dye was diluted with signal amplification reaction solution according to the manufacturer's instructions, and the DAPI was diluted with sterile water according to the manufacturer's instructions. The dilution ratio of the primary antibody and the corresponding dye are shown in Table 3.

[0075] Table 3. Preparation of primary antibody solution

[0076]

[0077] 3. Staining of sections:

[0078] The Leica Bond Rx automated staining platform was used for dewaxing, antigen retrieval, and multiple rounds of cyclic staining. After loading the slides into the instrument, the dewaxing program (see Table 4), antigen retrieval program (see Table 5), and staining program (see Table 6) were run in sequence.

[0079] Table 4 Dewaxing Procedure

[0080]

[0081]

[0082] Table 5 Antigen Repair Procedures

[0083]

[0084]

[0085] Table 6 Staining Procedure

[0086]

[0087]

[0088]

[0089]

[0090]

[0091]

[0092]

[0093]

[0094]

[0095]

[0096]

[0097]

[0098]

[0099]

[0100]

[0101] 4. Sealing:

[0102] Apply an appropriate amount of anti-fluorescence quenching mounting medium (Absin, included with the Abs50037 kit) to the tissue. Cover the tissue with a coverslip of appropriate size, ensuring even coverage and that the coverslip is flat on the tissue. Place the sealed section flat in a fume hood to air dry.

[0103] 5. Scanning Imaging:

[0104] The slices were mounted on the Akoya Vectra Polaris full-spectrum fluorescence imaging system for imaging.

[0105] Figure 1 This is a fluorescence map of the whole tissue region showing the co-expression of six biomarkers. Figure 2 The fluorescence image of the biomarker MIC A / B in the 480nm fluorescence channel. Figure 3 The fluorescence image of the biomarker ULBP4 in the 520nm fluorescence channel. Figure 4 The fluorescence image of the biomarker ULBP1 in the 570nm fluorescence channel. Figure 5 The fluorescence spectrum of the biomarkers ULBP2 / 5 / 6 in the 620nm fluorescence channel. Figure 6 The fluorescence image of the biomarker ULBP3 in the 700nm fluorescence channel. Figure 7 The fluorescence image of the biomarker CD56 in the 770nm fluorescence channel.

[0106] The above-described embodiments are merely preferred embodiments of the present invention. While the steps in these embodiments are detailed, they should not be construed as limiting the scope of the present invention. This embodiment uses a fully automated staining instrument, but it should not be considered that the scope of the present invention is limited to its use on fully automated staining instruments. For those skilled in the art, any substitutions made without departing from the scope of the claims of this patent are all within the protection scope of the present invention.

[0107] References

[0108] 1.Fuertes,MB,Domaica,CI,and Zwirner,NW(2021).Leveraging NKG2DLigands in Immuno-Oncology.Front.Immunol.12,1–27.https: / / doi.org / 10.3389 / fimmu.2021.713158.

[0109] 2. Liu, H., Wang, S., Xin, J., Wang, J., Yao, C., and Zhang, Z. (2019). Role of NKG2D and its ligands in cancer immunotherapy. Am.J. Cancer Res. 9, 2064–2078.

[0110] 3. Wensveen, FM V.,and B.(2018).NKG2D:A master regulator ofimmune cell responsiveness.Front.Immunol.9.https: / / doi.org / 10.3389 / fimmu.2018.00441.

[0111] 4.Zingoni,A.,Molfetta,R.,Fionda,C.,Soriani,A.,Paolini,R.,Cippitelli,M.,Cerboni,C.,and Santoni,A.(2018).NKG2D and its ligands:“One for all,all forone.”Front.Immunol.9.https: / / doi.org / 10.3389 / fimmu.2018.00476.

[0112] 5.Pang L,Ernst M,Huynh J.Development of a MultiplexImmunohistochemistry Workflow to Investigate the Immune Microenvironment inMouse Models of Inflammatory Bowel Disease and Colon Cancer.Int J MolSci.2021Oct 12;22(20):11001.doi:10.3390 / ijms222011001.PMID:34681666;PMCID:PMC8539370.

Claims

1. A kit for detecting ligands and CD56 of the NKG2D receptor, characterized in that, The kit comprises the following components: antibody eluent, primary antibody group, and multiplex fluorescent immunohistochemistry reagent group; the primary antibody group comprises specific antibodies against 6 biomarkers: ULBP1, ULBP2 / 5 / 6, ULBP3, ULBP4, MIC A / B, and CD56. The multiplex fluorescent immunohistochemical reagent set includes a set of TSA fluorescent dyes that correspond one-to-one with the specific antibodies. The antibody elution buffer is used for elution after staining with the specific antibody. The TSA fluorescent dye group includes: TSA480, TSA520, TSA570, TSA620, TSA700, TSA770 and DAPI; The TSA520 corresponds to the ULBP4 antibody; the TSA700 corresponds to the ULBP3 antibody; the TSA620 corresponds to the ULBP2 / 5 / 6 antibody; the TSA480 corresponds to the MIC A / B antibody; the TSA570 corresponds to the ULBP1 antibody; and the TSA770 corresponds to the CD56 antibody.

2. The reagent kit according to claim 1, characterized in that, The multiplex fluorescent immunohistochemistry reagent kit also includes a signal amplification reaction solution, a mouse-rabbit mixed HRP-labeled secondary antibody, and a blocking solution; And / or, the antibody elution buffer is an antibody elution buffer with an operating temperature of 37-50°C.

3. The reagent kit according to claim 2, characterized in that, The antibody elution buffer is Absin antibody elution buffer, catalog number Abs994.

4. The kit according to any one of claims 1-3, characterized in that, The kit also includes dewaxing solution, antigen retrieval solution, washing solution, 0.3%-5% hydrogen peroxide solution, ethanol solution, and anti-fluorescence quenching mounting medium.

5. The reagent kit according to claim 4, characterized in that, The hydrogen peroxide solution is a 3% hydrogen peroxide solution; and / or, the washing solution is a TBS washing solution.

6. The reagent kit according to claim 4, characterized in that, The antigen retrieval solution is an alkaline antigen retrieval solution with a pH value of 8.9-9.

1.

7. The reagent kit according to claim 6, characterized in that, The alkaline antigen retrieval solution is an EDTA solution.

8. A method for detecting ligands and CD56 of the NKG2D receptor for non-diagnostic purposes, characterized in that, The method uses the kit described in any one of claims 1-7 to stain FFPE sections after incubation with primary and secondary antibodies.

9. The method according to claim 8, characterized in that, The primary antibody incubation was performed sequentially using the anti-ULBP4, ULBP3, ULBP2 / 5 / 6, MIC A / B, ULBP1, and CD56 specific antibodies.

10. The method according to claim 8, characterized in that, The secondary antibody used for incubation was a hybrid HRP-labeled secondary antibody between mice and rabbits. And / or, the FFPE sections are washed with washing solution after incubation with the primary antibody and the secondary antibody, but before staining; And / or, the FFPE slices are FFPE slices that have undergone a sealing treatment; the sealing treatment is to incubate the FFPE slices with 0.3-5% hydrogen peroxide solution and then place them in a sealing solution for incubation at room temperature.

11. The method according to claim 10, characterized in that, The hydrogen peroxide solution is a 3% hydrogen peroxide solution.

12. The method according to claim 10, characterized in that, The FFPR slices that have undergone the blocking treatment are subjected to antigen retrieval treatment before incubation in 0.3-5% hydrogen peroxide solution.

13. The method according to claim 12, characterized in that, The hydrogen peroxide solution is a 3% hydrogen peroxide solution; and / or, the antigen retrieval treatment involves placing the FFPE slices in the antigen retrieval solution and retrieval at 98°C, followed by rinsing.

14. The method according to claim 12, characterized in that, The FFPE slides used for the antigen retrieval treatment are pretreated slides, or the FFPE slides used for the antigen retrieval treatment are dewaxed FFPE slides.

15. The method according to claim 14, characterized in that, The pretreatment consists of drying and dewaxing.

16. The method according to any one of claims 8-15, characterized in that, The method includes the following steps: Step 1: Place the slices in a 60℃ constant temperature oven and bake for 3 hours to obtain dried slices; Step 2: Place the dried sections in a dewaxing solution at 72°C for 1 minute, wash 3 times with ethanol solution, and wash 3 times with washing solution to obtain pretreated sections; Step 3: Place the pretreated slides in antigen retrieval solution at 98℃ for 20 minutes, and wash them 5 times with washing solution to obtain the retrieval slides; Step 4: Incubate the repaired slices in a 3% hydrogen peroxide solution at room temperature for 15 minutes, and wash them 3 times with the washing solution to obtain the hydrogen peroxide-treated slices. Step 5: Place the hydrogen peroxide-treated sections in a blocking solution at room temperature and incubate for 15 minutes to obtain the blocked sections; Step 6: Incubate the blocked sections with primary antibody solution at 37°C for 1 hour, and wash with washing buffer 4 times to obtain sections incubated with primary antibody. Step 7: After incubation with primary antibody, the slides are incubated with HRP-labeled secondary antibody (a hybrid of mouse and rabbit antibodies) at room temperature for 15-30 minutes, and then washed 4 times with washing buffer to obtain slides incubated with secondary antibody. Step 8: After incubation with secondary antibody, the sections are incubated with TSA fluorescent dye at room temperature for 15-30 minutes, and then washed 4 times with washing solution to obtain stained sections; Step 9: Incubate the stained sections in preheated antibody elution buffer at 37°C for 20 minutes, and wash 3 times with the washing buffer. Step 10: Repeat steps 5-9 four times, then repeat steps 5-8 once to label the six biomarkers and obtain six stained sections. Step 11: After 6 stainings, incubate the sections with DAPI at room temperature for 5-20 minutes, wash 3 times with washing buffer, and wash 3 times with sterile water to obtain the stained sections. Step 12: Mount the stained sections using an anti-fluorescence quenching mounting medium to obtain the mounted sections; Step 13: Use a full-spectrum fluorescence imaging system to photograph the slides after mounting, and obtain images of the fluorescence channels corresponding to each biomarker in the whole tissue slide and the merged fluorescence images.

17. The use of reagents for detecting NKG2D receptor ligands and CD56 in the preparation of a kit for detecting human breast cancer; said kit being the kit according to any one of claims 1-7.

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