CN116086919A

CN116086919A - Staining method and kit for lung cancer and/or pancreatic cancer samples

Info

Publication number: CN116086919A
Application number: CN202310136457.4A
Authority: CN
Inventors: 孙景云; 王光杓; 范师恒; 陈龙昀; 吴东方; 高志博
Original assignee: Shenzhen Yuce Biotechnology Co ltd
Current assignee: Shenzhen Yuce Biotechnology Co ltd
Priority date: 2023-02-20
Filing date: 2023-02-20
Publication date: 2023-05-09
Anticipated expiration: 2043-02-20
Also published as: CN116086919B

Abstract

The staining method comprises the steps of incubating lung cancer and/or pancreatic cancer samples to be detected sequentially by using corresponding primary antibodies according to the antigen sequence in the following antigen groups, and staining the incubated samples by using a staining agent to obtain stained samples: OX40, CD3, FOXP3, CD4, CD8, panCK. The invention can carry out multiple immunofluorescence experiments on a single slice, can simultaneously present the expression conditions of six antigen targets, and obviously improves the utilization rate of a single tissue sample, in particular to a precious sample.

Description

Staining method and kit for lung cancer and/or pancreatic cancer samples

Technical Field

The invention relates to the technical field of fluorescence detection, in particular to a method and a kit for staining lung cancer and/or pancreatic cancer samples.

Background

Lung cancer (Lung cancer) is a malignant tumor of the respiratory system that originates in the bronchus mucosa or glands of the Lung. The histopathology of lung cancer is divided into two major categories, non-small cell lung cancer and small cell lung cancer; has the characteristics of higher morbidity and mortality, and seriously threatens the life health and the quality of life of patients. The pathogenesis of the lung cancer is complex, and the lung cancer is influenced by factors such as environment, physics, chemistry, biological genetics and the like, so that clinical symptoms of early lung cancer patients are hidden, and most of lung cancer patients are transferred to late stage. At present, the treatment such as surgery, radiotherapy, chemotherapy and the like is mainly performed by directly killing tumor tissues, so that the body is greatly damaged. With the research of the lung cancer immunotherapy method, the research of developing tumor markers in cancer therapy has become an important point by killing tumor cells using the human autoimmune system. The tumor markers are substances generated by tumor cell synthesis, release or organism response to tumor cells in the tumor generation and proliferation process, can be detected from tissues, blood and body fluid, can indicate the existence of tumors when exceeding a normal detection threshold, develop different tumor markers, establish a rapid detection method and have great significance for accurately and efficiently diagnosing lung cancer and performing personalized treatment.

Immunostaining techniques include: IHC (immunochemistry), ICC (immunochemistry) and IF (Immunofluorescence). Wherein IHC/IF detection is performed by using antibodies and fluorescent detection to study the localization, relative expression and activation of target proteins in fixed cells or tissues in formalin-fixed and paraffin-embedded (formalin fixation and paraffin embedding, FFPE) samples. Today, the technology of tumor molecular detection is mature gradually, and the information base of related research and clinical decision is also beginning to pay more attention to the coverage of tumor microenvironment. The complexity of tumor microenvironments requires that a single Zhang Zuzhi sample slice yield multiple information, improving the utilization of the sample slice, particularly the precious sample. A recent new multi-labeled immunohistochemical staining/immunofluorescent staining (mhic/IF) technique allows simultaneous detection of multiple antibodies on a single tissue section and a thorough study of cell composition, cell function and cell-cell interactions. This technique overcomes many of the limitations of conventional immunohistochemical IHC techniques, including high variability between observers, with only one marker per tissue section being labeled. At present, the mIHC/mIF has been widely applied in domestic and foreign researches, and the technology is used for realizing the research on specific immune cell groups under tumor immune microenvironment (Tumor immune microenvironment, TIME), thereby being beneficial to the clinical prognosis judgment and the curative effect prediction of patients such as lung cancer and the like and becoming the technology with the most application prospect. The mIHC/mIF provides high-throughput multiple staining technology and standardized quantitative analysis for developing tissue research with high repeatability, high efficiency and high cost performance. The technology has great application value in transformation research and clinical practice, especially in tumor immunotherapy.

However, for lung cancer and pancreatic cancer samples, the existing methods have not been able to achieve multiplex immunofluorescent staining, i.e., detection.

Disclosure of Invention

According to a first aspect, in an embodiment, there is provided a method of staining a lung and/or pancreatic cancer sample, comprising:

the staining step comprises the steps of incubating lung cancer and/or pancreatic cancer samples to be detected sequentially by using corresponding primary antibodies according to the antigen sequence in the following antigen groups, and staining the incubated samples by using a staining agent to obtain stained samples:

OX40、CD3、FOXP3、CD4、CD8、panCK。

according to a second aspect, in an embodiment, there is provided a sample or image obtained by the staining method of any of the first aspects.

According to a third aspect, in an embodiment, there is provided an image analysis method including: and (3) carrying out biomarker analysis calculation on the fluorescence expression data of the target area of the image in the second aspect to obtain a biomarker value of the sample.

According to a fourth aspect, in an embodiment, there is provided a kit for detecting a lung cancer and/or pancreatic cancer sample, comprising the following antibodies:

anti-OX 40 monoclonal antibody, anti-CD 3 monoclonal antibody, anti-FOXP 3 monoclonal antibody, anti-CD 4 monoclonal antibody, anti-CD 8 monoclonal antibody, anti-panCK monoclonal antibody.

According to the method and the kit for staining lung cancer and/or pancreatic cancer samples, disclosed by the embodiment of the invention, multiple immunofluorescence experiments can be carried out on a single slice, the expression condition of six antigen targets can be simultaneously presented, and the utilization rate of a single tissue sample, especially a precious sample, is obviously improved.

In one embodiment, the method and kit of the invention can be used for analyzing TILs of lung cancer and pancreatic cancer immune microenvironment, and have excellent accuracy and precision.

In one embodiment, the invention facilitates the observation of interactions and symbiotic localization of target proteins, with significantly reduced demand for tissue samples; and can be applied to automatic dyeing machine, show improvement degree of accuracy and repeatability, simple and convenient high efficiency.

Drawings

FIG. 1 is a schematic diagram of the detection flow in example 1;

fig. 2.1 is a CD3 antibody: staining results plot of antibody dilution = 1:500;

fig. 2.2 is a CD3 antibody: staining results plot of antibody dilution = 1:800;

fig. 2.3 is a CD3 antibody: staining results plot of antibody dilution = 1:1000;

FIG. 3.1 shows IHC staining results for CD3 antibody repair;

FIGS. 3.2 to 3.4 are graphs showing staining results of antibody repair at 1 st, 3 rd and 5 th times, respectively;

FIG. 4 shows MIF and SNR (signal to noise ratio) results for different repair times of CD3 antibodies;

FIG. 5 shows dye-paired MIF and SNR (signal to noise ratio) results for CD4 antibodies;

FIG. 6 shows MIF and SNR results for various repairs of CD4 antibodies;

FIG. 7 is a dye-paired MIF and SNR results for CD8 antibodies;

FIG. 8 shows MIF and SNR results for various repairs of CD8 antibodies;

FIG. 9.1 is a graph showing the staining results of 21R5148SLZB tonsil samples;

FIG. 9.2 is a graph showing staining results of 21R7214SLZB lymph node samples;

FIG. 10.1 shows IHC staining results for OX40 antibody dye pair;

FIGS. 10.2-10.4 are staining results for

channels

480, 620, 690, respectively, of an OX40 antibody;

FIG. 10.5 is MIF and SNR results of OX40 antibody dye pairing;

FIG. 11.1 shows IHC staining results for OX40 antibody repair;

FIGS. 11.2 to 11.4 show staining results of antibody repair at 1 st, 3 rd and 5 th times, respectively;

FIG. 11.5 is MIF and SNR results for different number of repairs of OX40 antibodies;

FIG. 12 is a FOXP3 dye paired MIF and SNR results;

FIG. 13 shows MIF and SNR results for various repairs of FOXP3 antibodies;

FIG. 14 shows MIF and SNR results of panCK dye pairing;

FIG. 15 shows MIF and SNR results for various repairs of panCK antibodies;

FIG. 16 shows the staining results of tonsil samples (21R 5148 SLZB);

FIG. 17 shows the mIF condition staining results of tonsil sample 21R5148 SLZD-1;

FIG. 18 shows the mIF conditioned staining results of pancreatic cancer samples 2104370 FZZZA-11;

FIG. 19 shows the mIF condition staining results for lung cancer samples 2100996 FZZZA;

FIG. 20 is the result of staining (20X) of lung cancer samples;

FIG. 21 shows the results of positive cell density correlation analysis of IHC and mIF detection in lung and pancreatic cancer samples;

FIG. 22 shows the results of a positive cell proportion correlation analysis of IHC and mIF detection in lung and pancreatic cancer samples;

FIG. 23 is the staining results (200X) of repeated experiments in lung and pancreatic cancer samples.

Detailed Description

The invention will be described in further detail below with reference to the drawings by means of specific embodiments. In the following embodiments, numerous specific details are set forth in order to provide a better understanding of the present application. However, one skilled in the art will readily recognize that some of the features may be omitted in various situations, or replaced by other materials, methods. In some instances, some operations associated with the present application have not been shown or described in the specification to avoid obscuring the core portions of the present application, and may not be necessary for a person skilled in the art to describe in detail the relevant operations based on the description herein and the general knowledge of one skilled in the art.

Furthermore, the described features, operations, or characteristics of the description may be combined in any suitable manner in various embodiments. Also, various steps or acts in the method descriptions may be interchanged or modified in a manner apparent to those of ordinary skill in the art. Thus, the various orders in the description and drawings are for clarity of description of only certain embodiments, and are not meant to be required orders unless otherwise indicated.

The numbering of the components itself, e.g. "first", "second", etc., is used herein merely to distinguish between the described objects and does not have any sequential or technical meaning. The terms "coupled" and "connected," as used herein, are intended to encompass both direct and indirect coupling (coupling), unless otherwise indicated.

Herein, "room temperature" means 23±2 ℃, also called normal temperature.

CD3: the alias OKT3, whole T cell markers, immunoglobulin superfamily members, common antibodies recognizing T cells, complexes consisting of delt, epsilon, gamma, zeta and like chains. The application in pathology is diagnosis of T cell lymphoma, which is the most specific antibody of T cells, and the signal is localized in the cell membrane and cytoplasm. CD3, a T cell surface marker, has a primary function of promoting activation of cytotoxic T cells (cd8+ T cells) and helper T cells (cd4+ T cells) and ultimately helping to develop an adaptive immune response.

CD4: alias OKT4, helper T cell markers. Most macrophages, follicular dendritic cells, langerhans cells were also positive. The application in pathology is the diagnosis and classification of T cell lymphocytes, with signals localized to the envelope. CD4 and CD4+ T cells belong to helper T cells in lymphocyte classification, directly reflect the intensity of the immunity function of an organism, and are main targets in the HIV infection process.

CD8: is a leukocyte differentiation antigen used to assist T Cell Receptor (TCR) in recognizing antigen and involved in T cell activation signal transduction. T cells expressing CD8 (cd8+ T cells) typically differentiate into cytotoxic T cells upon activation, capable of specifically killing target cells, and signals are localized to the envelope. Cd8, cd8+ T cells, cytotoxic T Lymphocytes (CTLs), secrete a variety of cytokines specifically to participate in the immune function. Has killing effect on some virus, tumor cell and other antigen matter and forms important antiviral and antitumor immunity defense line with natural killer cell.

FOXP3: the major regulator of Treg, foxp3, is one of the key transcription factors controlling Treg cell development and function. In addition to helping the body avoid autoimmune diseases, the immunosuppression of Treg cells can also, in some tumors, make the body antigen-tolerant to tumor cells, thereby allowing tumor cells to escape from the body's immune killing. Regarding how to perform detection analysis on Treg cells, currently, the well-known surface markers for differentiating Treg cells are: cd4+cd25+foxp3+ or cd4+cd25+cd127. FoxP3 is a necessary factor for the development and maturation of Treg cells and functions, is the most sensitive marker of Treg cells at present, but detection of FoxP3 requires rupture of membranes of cells, such as the need for loading cells in subsequent experiments and the cells remain intact, and can be used as Treg markers by low expression of cd4+cd25+cd127, with CD127 on the surface of Treg cells.

OX40: OX40 (TNFRSF 4, CD 134) is a member of the Tumor Necrosis Factor (TNF) receptor superfamily, which can regulate T cell activity and immune response. OX40 protein comprises four cysteine-rich domains, one transmembrane domain and one cytoplasmic tail containing the QEE motif. OX40 is expressed predominantly in activated cd4+ and cd8+ T cells, whereas OX40 ligand (OX 40L, TNFSF4, CD 252) is expressed predominantly in activated antigen presenting cells. OX40 binding to OX40L results in recruitment of TNF receptor-related factors (TRAFs) to form TCR-independent signaling complexes. One component of the complex (PKCθ) activates the NF- κB pathway. OX40 signaling by Akt also directly enhances TCR signaling. Studies have shown that the OX40L-OX40 pathway is associated with inflammation and autoimmune diseases. Other studies have shown that OX40 agonists enhance anti-tumor immunity in a variety of cancer types. Immune activation of OX40 with tumors in the tumor microenvironment can result in OX40 expression. Can enhance the activation and proliferation of effector T cells and make Tregs, thereby leading to complex anti-tumor immune responses. Tumor infiltrating T cells express OX40 and injection of Anti-OX40 antibodies or OX40l.fc fusion proteins are effective in inhibiting tumor growth in tumor-bearing mouse models. Currently, a variety of Anti-OX40 antibodies or OX40l.fc fusion proteins are being used in clinical trials against metastatic cancers. To increase the effect of anti-OX40, combining the OX40 pathway with other, possibly complementary, immune pathways at the appropriate time may be a key strategy for more effective activation of anti-tumor immune responses, and many studies on different combination therapies are currently underway.

panCK: cytokeratin is a group of at least 29 different proteins, which is characteristic of epithelial cells and villus cells.

Cytokeratins

1, 4, 5, 6 and 8 are members of the neutral to basic subfamily type II. Monoclonal anti-cytokeratin is a specific marker of epithelial cell differentiation and has been widely used for identification and classification of tumors. Monoclonal anti-ubiquitin is a broadly reactive agent that recognizes epitopes present in most human epithelial tissues. It facilitates the typing of normal cells, metaplastic cells and tumor cells. Synergy between the different components results in staining amplification. This makes it possible to identify cells which would otherwise be stained only in small amounts. This mixture helps to distinguish between cancerous and non-epithelial tumors, such as sarcomas, lymphomas, and neural tumors. It also helps to detect micrometastases in lymph nodes, bone marrow and other tissues and to determine the origin of poorly differentiated tumors. Cytokeratins are of two types, acidic type I cytokeratins and basic or neutral type II cytokeratins. Cytokeratins are typically present in pairs, including type 1 cytokeratins and type 2 cytokeratins. Typically, type II cytokeratins are 8kD larger than type I cytokeratins. The signal is localized to the cytoplasm.

OX40 is expressed predominantly in activated cd4+ and cd8+ T cells; foxP3 is a necessary factor for the development and maturation and function of Treg cells, and is the most sensitive marker of Treg cells at present; CD4 is a helper T cell marker; CD8 is a leukocyte differentiation antigen used to assist T Cell Receptors (TCRs) in recognizing antigens and involved in the transduction of T cell activation signals; CD3 is a whole T cell marker, an immunoglobulin superfamily member, a common antibody that recognizes T cells; panCK is an epithelial-derived tumor marker.

The present invention provides a mIF Panel for detecting T cell immune checkpoints in tumor tissue; and meanwhile, antibody development is carried out, and finally, the target marker is confirmed to be: CD3, CD4, CD8, OX40, FOXP3, panCK.

OX40, CD3, FOXP3, CD4, CD8, panCK. The sample may be a sample taken from a subject who has lung cancer alone, a sample taken from a subject who has pancreatic cancer alone, or a sample taken from a subject who has both lung cancer and pancreatic cancer.

The staining method gives a stained sample which is an intermediate result, not a final result, and therefore not a diagnostic method for a disease.

In one embodiment, the number of types of staining agents used in the staining step corresponds to the number of types of antibodies.

In one embodiment, in the staining step, the primary antibody corresponding to the antigen and the staining agent are paired and stained in sequence as follows:

anti-OX 40 monoclonal antibody paired Opal 690, anti-CD 3 monoclonal antibody paired Opal 620, anti-FOXP 3 monoclonal antibody paired Opal 570, anti-CD 4 monoclonal antibody paired Opal 520, anti-CD 8 monoclonal antibody paired Opal 480, anti-panCK monoclonal antibody paired Opal 780.

In one embodiment, the staining step comprises repeating antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, staining (i.e., dye incubation), and antibody removal in the order of antigen. Each round carries out antigen repair, blocking, primary antibody incubation, secondary antibody incubation, staining and antibody removal on one antigen.

In one embodiment, the secondary antibody used during the secondary antibody incubation is labeled with horseradish peroxidase in the staining step.

In one embodiment, in the staining step, after staining the first 5 antigens, the sample is blocked by using a blocking solution, and then the 6 th antigen is sequentially subjected to primary antibody incubation, secondary antibody incubation, signal amplification staining, antibody removal, staining by using a corresponding stain, and staining by using a nuclear stain, so as to obtain a stained sample.

In one embodiment, the stain used in the signal amplification staining step comprises Opal TSA-DIG dye.

In one embodiment, the nuclear stain includes, but is not limited to, DAPI (i.e., 4', 6-diamidino-2-phenylindole, CAS registry number 47165-04-8).

In one embodiment, in the staining step, the sample comprises a cancer tissue section.

In an embodiment, the method further comprises an imaging step, including performing continuous spectrum imaging on the dyed sample to obtain a corresponding dyed image. The stained image is an intermediate result, not a final result, and therefore, is not a diagnostic method of a disease.

In one embodiment, the method further comprises a detection step of detecting the stained image. The test result may generally be an analysis result of biomarker values, which is merely an intermediate result, not a final result, and thus is not a diagnostic method for a disease.

In one embodiment, a staining agent is further included, and the number of kinds of staining agent is consistent with the number of kinds of antibody.

In one embodiment, the stain comprises a fluorescent stain.

In one embodiment, the stain comprises:

Opal 690、Opal 620、Opal 570、Opal 520、Opal 480、Opal 780。

in one embodiment, the pairing relationship of the antibody and the staining agent is as follows:

In one embodiment, the fluorescent dye Opal TSA-DIG is also included.

In one embodiment, the kit further comprises at least one of an antigen retrieval solution, a horseradish peroxidase-labeled secondary antibody, a blocking solution, and an anti-fluorescence quencher.

In one embodiment, a fully automated multicolor immunofluorescence assay kit and staining method for lung and pancreatic cancer samples is provided.

In one embodiment, the kit provided by the invention comprises the following components: peroxidase blocking agent, primary antibody reagent, secondary antibody reagent and fluorescent dye; the combined primary antibody comprises a rabbit anti-human CD3 monoclonal antibody, a rabbit anti-human CD4 monoclonal antibody, a mouse anti-human CD8 monoclonal antibody, a rabbit anti-human OX40 monoclonal antibody, a rabbit anti-human FOXP3 monoclonal antibody and a rabbit anti-human panCK monoclonal antibody; the combined secondary antibody reagent comprises an HRP (horseradish peroxidase) labeled anti-mouse and anti-rabbit mixture. The kit can simultaneously present the expression conditions of six antigen targets on one tissue sample slice, is convenient for observing the interaction and symbiotic positioning of target proteins, and remarkably reduces the demand on the tissue samples; the kit can be applied to an automatic dyeing machine, and is simple and efficient, and accuracy and repeatability are remarkably improved.

In one embodiment, a fully automated multicolor immunofluorescence assay kit and staining method for lung and pancreatic cancer samples is provided. The invention develops a detection kit through a multiple immunofluorescence technology, and utilizes an image recognition system (such as an InFormv 2.3 and a HALO pathology image analysis system) to obtain tissue and cell phenotype information, fluorescence intensity information of corresponding targets and the like, so as to analyze TILs of lung cancer and pancreatic cancer immune microenvironments, verify the detection accuracy and precision of the kit, and meet scientific research detection requirements. The invention mainly solves the technical problems that the tumor immunotherapy efficacy evaluation lacks enough biomarkers and how to evaluate the immunotherapy efficacy in a larger scale by related indexes of tumor immune microenvironment; meanwhile, the requirement of tumor immune microenvironment analysis by a multiple immune fluorescence technology in clinical pathology work and scientific research is well met, and doctors and scientific researchers are efficiently assisted to complete the analysis of various immune histochemical indexes after immune histochemical multiple marking.

In one embodiment, the kit can simultaneously express six antibodies on one tissue section, and can provide more target protein information and co-localization conditions for pathologists, so that the staining efficiency can be greatly improved, and samples can be saved; meanwhile, the problems of low repeatability, long time and the like caused by manual dyeing can be avoided; the kit can be applied to a full-automatic sheet dyeing machine and has wide application prospect.

Example 1

The embodiment provides a full-automatic multicolor immunofluorescence detection kit for lung cancer and pancreatic cancer samples, which comprises the following components: peroxidase blocking agent, primary antibody reagent, secondary antibody reagent and fluorescent dye; the combined primary antibody comprises a rabbit anti-human CD3 monoclonal antibody, a rabbit anti-human CD4 monoclonal antibody, a mouse anti-human CD8 monoclonal antibody, a rabbit anti-human OX40 monoclonal antibody, a rabbit anti-human FOXP3 monoclonal antibody and a rabbit anti-human panCK monoclonal antibody; the combination secondary antibody reagent comprises an HRP-labeled anti-mouse and anti-rabbit mixture. The key materials involved in the full-automatic multicolor immunofluorescence detection are shown in Table 1.

TABLE 1 Key bill of materials

Obtaining lung cancer and pancreatic cancer tissue samples from each subject, preparing tissue sections, performing multiple immunohistochemical staining treatment (such as Bond RX automatic staining instrument of Leica company) on the lung cancer and pancreatic cancer tissue samples, and obtaining corresponding immunohistochemical microscopic panorama by an imaging scanning instrument (such as Vectra polar is spectral type quantitative pathological analysis system of Akoya company).

The method is suitable for a full-automatic immunohistochemical staining machine, and the staining procedure is shown in the following table.

Table 2 6 mIF staining procedure for dyes

Table 3 6 dyeing procedure for mIF yin-ginseng samples with dyes

The accuracy and the precision of the embodiment are qualified through a performance verification experiment. Sample H by pathologist&E, judging a dyeing result, wherein a sample meets the quality control requirement; the staining results revealed that subcellular localization was correct (CD 3, CD4, CD8, OX40 signals were localized to the cell membrane, panCK signals were localized to the cytoplasm, FOXP3 signals were localized to the nucleus); the accuracy verification result shows that the density of positive cells and the proportion correlation coefficient are both larger than 0.6, and the acceptance criterion is met. The results of the precision verification show that when the positive cell Density (Overall Density) is greater than 100/mm ² The c.v. average distribution is about 0.08 to 0.2; when the positive cell density is less than 100/mm ² The c.v. average distribution is about 0.17 to 0.38, meeting acceptance criteria.

The present invention will be further described with reference to specific embodiments, but the present invention is not limited to the examples of lung cancer and pancreatic cancer, and the lung cancer and pancreatic cancer are selected for performance verification experiments only during development.

As shown in fig. 1, a schematic diagram of a detection flow is shown, and specific steps are as follows:

1. full-automatic multicolor immunofluorescence detection kit for preparing lung cancer and pancreatic cancer samples

The embodiment provides a full-automatic multicolor immunofluorescence detection kit for lung cancer and pancreatic cancer samples, and the development process is as follows:

in the methodology development experiments, each antibody was optimized. The antibody comprises: CD3, CD4, CD8, OX40, FOXP3, panCK.

The early stage of antibody test and experimental condition fumbling, including links such as preliminary verification of antibody characteristics, fumbling of antibody concentration, pairing of antibody dyes, fumbling of antibody repair times, preliminary experiments of mIF experiments, and the like, finally determines specific antibodies and experimental conditions.

The determination of the mIF condition includes: co-dyeing, channel selection, dyeing round selection and overall dyeing effect.

The method comprises the following specific steps:

(1) Antibody validation experiments: using IHC test to confirm whether the antibody can be used; and primarily determining a ginseng sample;

(2) Concentration resistance fumbling experiments: and (3) carrying out proper concentration adjustment by combining the concentration recommended by the antibody specification, setting 2-3 concentrations, and developing an IHC experiment to find the primary antibody concentration. For example: antibody Anti-CD68 (KP 1) -Abcam, recommended antibody concentration of 1:100, 1 may be set: 50. 1: 100. 1:200, performing IHC experiments;

(3) Antibody acid-base repair experiments: and (3) confirming ER1 or ER2 by combining with the repair conditions recommended by the antibody instruction, carrying out IHC experiments based on the common conditions of 95 ℃ for 20min, and adjusting according to the experimental results. For example: adjusting ER1 to ER2; the condition parameters can be adjusted to 95 ℃ for 20min;95 ℃ for 40min;95℃for 80min.

(4) Antibody dye pairing experiments: panel (CD 3, CD4, CD8, OX40, FOXP3, panCK) is a six-label seven-color mIF assay, planned to use

channels

480, 520, 570, 620, 690, 780, paired channels are recommended in conjunction with antibody specifications and related references, and 3-7 channel antibody dye paired IF assays are performed on different antibodies; and (3) carrying out fluorescence intensity statistics on experimental results, calculating a signal to noise ratio, and confirming a better pairing channel of the antibody. For example: antibody Anti-CD68 (KP 1) -Abcam, can be set to carry out IF pairing experiments with 7 different channels of 480, 520, 570, 620, 650, 690, 780 and the like, and the results show that the signal to noise ratio of the channels of 650, 780 and 570 is higher, and in the subsequent mIF experiment condition optimization, the 3 channels are preferably selected to carry out the pairing of the antibody dye.

In the early-stage experiment, the dyeing sequence is firstly fumbling the performance of the repairing times of the antibody, and the antibody which shows better dyeing effect for less repairing times is firstly dyed; for antibodies exhibiting better staining effect for more repair times, post staining in panel; wherein the signal to noise ratio is calculated as a measurement basis. In addition, the dye condition is finally determined by debugging for a plurality of times in the mIF condition fumbling.

For dyeing pairing selection, in the process of antibody development, 3-8 different dyeing channel IF tests are carried out, and the priority order of channel selection is measured by calculating the signal-to-noise ratio; and then, carrying out multiple adjustment in the subsequent mIF experiment, avoiding cross color, better showing the positive dyeing effect and determining the dyeing condition.

(5) Antigen retrieval frequency experiment: panel (CD 3, CD4, CD8, OX40, FOXP3, panCK) is a six-label seven-color mIF experiment, antigen retrieval is 7 times, and DAPI staining is the last time; performing IF experiments after performing antigen repair on different antibodies for 1-6 times, and selecting 1, 3 and 5 rounds for repair experiments; and (3) carrying out fluorescence intensity statistics on experimental results, calculating a signal to noise ratio, and confirming the superior repair turn of the antibody. For example: antibody Anti-CD68 (KP 1) -Abcam, setting 1, 3, 5 times of repair and then incubating the primary antibody for performing the IF experiment of repair times, and arranging the antibody in the optimal repair time to perform the experiment according to the signal-to-noise ratio result and the subsequent mIF experiment condition optimization.

(6) mIF pre-experiment: summarizing the antibody characteristics according to the experimental results, determining primary antibody concentration, acid-base repair conditions, antibody pairing dyes and antigen repair rounds, and primarily determining mIF experimental conditions; and optimizing mIF conditions according to experimental results until the effects of no nonspecific dyeing, no cross-dyeing after spectrum resolution and the like are achieved.

A. The screening procedure for each antibody was as follows:

cd3 antibody concentration: the antibody is recombinant Anti-CD3 epsilon antibody [ CAL54] (ab 237707) -Abcam.

Dilution ratio of antibody fuzzing: fig. 2.1 is a CD3 antibody: staining results for antibody dilutions = 1:500, figure 2.2 is a CD3 antibody: staining results for antibody dilutions = 1:800, figure 2.3 is a CD3 antibody: staining results plot of antibody dilution=1:1000.

Number of antibody repairs: 21R4111SLZA, staining results are shown in FIGS. 3.1-3.4, FIGS. 3.2-3.4 are staining results of antibody repair 1 st, 3 rd, 5 th, MIF and SNR (signal to noise ratio) results are shown in Table 4 and FIG. 4, respectively.

TABLE 4 Table 4

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					CD3	1 time, 3 times, 5 times: 58.72	5 times>3 times>1 time	5 times>3 times>1 time	Can all be

Cd4 antibody concentration: the antibody is recombinant Anti-CD4 antibody [ EPR6855] (ab 133616) -Abcam, and the dilution ratio is 1: 500. 1: 1000. 1:2000.

CD4 dye pairing: MIF and SNR results are shown in table 5 and fig. 5.

TABLE 5

CD4 antibody repair times: MIF and SNR results are shown in table 6 and fig. 6.

TABLE 6

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					CD4	226.17 for 1 time, 24.47 for 3 times, 5 times: 25.71	3 times>5 times	3 times>5 times	3

Cd8 antibody concentration:

CD8 Monoclonal Antibody (4B 11) (MA 1-80231) -Thermo Fisher Scientific, dilution ratio 1:50, 1: 100. 1:200.

Recombinant Anti-CD8 alpha antibody [ EP1150Y ] (ab 93278) -Abcam, dilution ratio of 1: 2000. 1: 4000. 1:8000.

CD8 dye pairing: MIF and SNR results are shown in table 7 and fig. 7.

TABLE 7

CD8 antibody repair times: MIF and SNR results are shown in table 8 and fig. 8.

TABLE 8

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					CD8	1 time 4.23, 3 times 5.54, 5 times: 3.3	5 times>3 times>1 time	5 times>3 times>1 time	3、5

Ox40 antibody concentration: 21R5148SLZB tonsils and 21R7214SLZB lymph node samples were selected.

The incubation conditions were as follows:

TABLE 9

Fig. 9.1 shows the staining results of 21R5148SLZB tonsil samples, and fig. 9.2 shows the staining results of 21R7214SLZB lymph node samples, which shows that the staining effect is good, and the antibody dye pairing experiment can be performed.

OX40 antibody dye pairing: 21R5148SLZB tonsil samples. Fig. 10.1 shows IHC staining results, and fig. 10.2 to 10.4 show staining results of

channels

480, 620, 690, respectively. Fig. 10.5 shows MIF and SNR results.

Table 10

Antibodies to	Exposure time (ms)	MIF	SNR	Recommendation
					OX40	480:7.52、620:38.11、690:24.76	480>690>620	620>690>480	480>690>620

Number of OX40 antibody repairs: 21R5148SLZB tonsil samples. Fig. 11.1 shows IHC staining results, and fig. 11.2 to 11.4 show staining results of antibody repair 1 st, 3 rd, and 5 th times, respectively. Fig. 11.5 shows MIF and SNR results.

TABLE 11

Antibodies to	Exposure time (ms)	MIF	SNR	Recommendation
					OX40	1 time, 3 times, 5 times: 7.6	3 times>5 times>1 time	3 times>1 time>5 times	Can all be

Foxp3 antibody concentration:

FoxP3 (D6O 8R) Rabbit mAb (12653S) -Cell Signaling Technology, dilution ratio was 1:800.

Anti-FOXP3 Anti-body [ EPR22102-37] (ab 215206) -Abcam, dilution ratio is 1: 25. 1: 50. 1:250.

FOXP3 dye pairing: fig. 12 shows MIF and SNR results.

Table 12

Antibodies to	Exposure time (ms)	MIF	SNR	Recommendation
					FoxP3	480:5.01、570、690:54.57	480>670>690	480>570>690	480、570

FOXP3 antibody repair times: fig. 13 shows MIF and SNR results.

TABLE 13

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					FoxP3	1 time 70.8, 3 times 69.8, 5 times 69.8	5 times>3 times>1 time	3 times>5 times>1 time	3>5

Panck antibody concentration:

anti-pan Cytokeratin antibody [ KRT/1877R ] (ab 234297) -Abcam, dilution ratio 1: 200. 1:400.

pan-Cytokeratin antibody (AE 1/AE 3) (sc-81714) -Santa Cruz Biotechnology, dilution ratio 1: 300. 1: 600. 1:900.

pairing panCK dyes: fig. 14 shows MIF and SNR results.

TABLE 14

Antibodies to	Exposure time (ms)	MIF	SNR	Recommendation
					CD4	480:6.65、520:20.56、650:7.07	620>650>690>480	650>480>690>620	(650)480

Number of panCK antibody repairs: fig. 15 shows MIF and SNR results.

TABLE 15

Antibodies to	570 exposure time (ms)	MIF	SNR	Recommendation
					CD56	54.72 times, 3 times 29.84 times, 5 times: 39.52	5 times>3 times>1 time	3 times>5 times>1 time	Can all be

B. mIF Pre-experiment

mIHC pre-experiment plan-pre-experiment 1.

Table 16

mhic-pre-experiment 1 results: tonsil samples (21R 5148 SLZB), the staining results are shown in FIG. 16.

mIF performance validation experiment-sample validation:

confirming pancreatic cancer samples for performance verification experiments:

TABLE 17

Performance verification experiment sample arrangement scheme:

repeatability: 2 pancreatic cancer samples, specifically 2204204FZZA, 2104370FZZA.

Accuracy: 5 pancreatic cancer samples, specifically 2204204FZZA, 2104370FZZA, 2109096FZZA, 2010613FZZA, 22R6152SLZA.

Confirming lung cancer samples for performance verification experiments:

TABLE 18

Performance verification test sample:

repeatability: 2 lung cancer samples, specifically 22R2897SLZA, 22R3184SLZA.

Accuracy: the 5 lung cancer samples are specifically 22R2897SLZA, 22R3184SLZA, 22R4221SLZA, 22R3149SLZA and 22R7458SLZA.

And (3) performing automatic dyeing by utilizing BOND RX, performing multiple mIF condition optimization, and finally determining a dyeing program as shown in the table below.

Table 19 experimental parameters

mIF condition (Table 19): tonsil sample 21R5148SLZD-1, and the staining results are shown in FIG. 17.

mIF condition (Table 19): pancreatic cancer sample 2104370FZZA-11, staining results are shown in FIG. 18.

Results of mif pre-experiments: the staining results of lung cancer sample 2100996FZZA are shown in fig. 19. The results show that 780 does not develop color; pre-experiment adjustment: the samples were replaced.

2. Application of the kit in a full-automatic sheet dyeing machine and a dyeing program. The method comprises the following steps:

the kit is applied to lung cancer and pancreatic cancer tissues, tonsil tissues are taken as yang ginseng, multiple immunohistochemical staining is implemented, and a machine used is a BOND RX full-automatic sheet dyeing machine.

(1) Taking lung cancer, pancreatic cancer and tonsil paraffin tissue, respectively preparing 1 paraffin tissue slice (experimental slice) for lung cancer and pancreatic cancer, 2 paraffin tissue slices (Ginseng radix and Yang Can slice) for tonsil paraffin tissue, attaching sample labels with the thickness of 3 micrometers, and placing in a slice box for standby in a refrigerator at the temperature of 4 ℃;

(2) And opening the electrothermal blowing drying box, and preheating the electrothermal blowing drying box to 65 ℃ in advance. Placing the slices into a slice rack, and placing the slice rack into an electrothermal blowing drying oven at 65 ℃ for 3 hours. And (3) observing the wax on the surface of the tissue by naked eyes to thoroughly melt, and filling in an experiment record table.

(3) Reagent preparation: taking out the primary antibody, the secondary antibody, the dye and the sealing liquid from the refrigerator at the temperature of 4 ℃, wrapping a test tube with tinfoil paper for the reagent needing to be protected from light, placing the test tube on ice for standby, and placing the test tube in a centrifuge for short centrifugation.

a. Antibody preparation: taking out the antibody stock solution, and placing the antibody stock solution in a centrifugal machine for short centrifugation for later use. In a burette (Titration Kit), working solution was prepared from antibody diluent according to the dilution ratio in the following table, a test tube was wrapped with tinfoil paper, the dye name was marked outside the tube, and the preparation time, and the preparation personnel.

Table 20 preparation of working solution for primary antibody

b. Dye preparation:

(1) preparing dye storage liquid, taking out antibody and Opal dye, wrapping a test tube with tinfoil paper, and placing in a centrifuge for short centrifugation. Add 75. Mu.L of DMSO solution to each of Opal 480, opal 520, opal 570, opal 620, opal 690, TSA-DIG dye tubes; 300 μl of deionized water solution was added to the Opal 780 dye tube. After the dye tube is uniformly mixed up and down for 10 times, the dye tube is put into a centrifuge for short centrifugation after the reagent is completely dissolved.

(2) Taking out the dye storage liquid, and putting the dye storage liquid into a centrifugal machine for short centrifugation for standby. In a burette (Titration Kit), a working solution was prepared from a diluent in accordance with the dilution ratio shown in the following table, a test tube was wrapped with tinfoil paper, the dye name was marked outside the tube, and the preparation time was measured.

Table 21 dye working solution formulation

c. Preparing a secondary antibody: the secondary antibody was pipetted into a burette (calculation formula for the amount of secondary antibody reagent: 1618+150×7n (μl), where n is the number of sections, 1618 μl is the dead cartridge volume of 30mL of the kit, and 150 μl is the amount per section).

d. Preparing a sealing liquid: the blocking solution was pipetted into a burette (formula for blocking solution usage: 1618+150×7n (μl), where n is the number of slices, 1618 μl is the dead cartridge volume of 30mL of the kit, and 150 μl is the usage per slice).

(4) Setting a dyeing program:

a. detecting samples and setting a yang ginseng sample program: clicking on "program settings" see table below for specific parameters.

Tables 22 KN026 208 Opal 6-color mIF protocol

b. Setting a yin ginseng sample program: clicking on "program settings" see table below for specific parameters.

Table 23 procedure for staining mIF yin-ginseng samples for 6 dyes

(5) The detection system and the reagent rack filled with the antibody and the dye are put into a BOND RX instrument, and a dyeing program is started:

(6) The right-click slide rack directly starts to dye, and the dyeing start time and the dyeing end time are displayed below the slide rack, so that an experiment record table is filled.

(7) Sealing piece:

a. after dyeing is finished, a 'loading/unloading button' below the slide frame is pressed, the locking state of the slide frame is released, the slide frame is taken out, the primary antibody and the secondary antibody box are sealed by a sealing film or a cover, the primary antibody and the secondary antibody box are put into a refrigerator at the temperature of 4 ℃ for preservation, TSA-DIG, opal780 and DAPI dyes are directly poured into a waste liquid bottle, and other Opal dyes can be continuously used in the working solution validity period.

b. Sections were removed from the slide rack and the universal slide cover was removed (Bond Universal Covertiles). Except for the tissue, the water trace on the slice was wiped off with dust free paper.

c. 1-2 drops of the anti-fluorescence quenching agent are dripped into each slice for sealing, so that the anti-fluorescence quenching agent is ensured to cover tissues without bubbles, and meanwhile, the anti-fluorescence quenching agent needs to be prevented from overflowing. And (3) placing the slices in a storage plate for storage, keeping the slices away from light at room temperature for 30min, and placing the slices in a refrigerator at 4 ℃ for keeping the slices away from light for scanning after the anti-fluorescence quenching agent is solidified.

(8) Scanning slice: the slices were scanned in full slices using a Vectra polar instrument, setup procedure. After the end of the scan, the positive control and negative control samples were ensured to have the expected staining effect. Positive control expected staining effect: positive signals appear in the samples (cell membrane localization including CD3, CD4, CD8, OX40, cytoplasmic localization panCK, nuclear localization FOXP3, no obvious cross-color, uniform staining). Negative control expected staining effect: no significant positive signal was seen with the samples. FIG. 20 shows the result of staining (20X) of lung cancer samples. FIG. 18 shows the staining results (20X) of pancreatic cancer samples.

(9) Data analysis: the data is uploaded to the shared folder, and the path is the designated project folder. And then data processing is carried out according to analysis requirements.

3. Performance verification experimental methods and results of the kit applied to lung cancer and pancreatic cancer tissues. The specific experiment is as follows:

(1) Sample:

a. to evaluate the Accuracy of polychromatic immunofluorescence results (Accuracy), 5 FFPE samples from lung and pancreatic cancer each were tested, each of which was selected for performance verification by 10 serial sections, 6 for IHC monochromatic immunohistochemistry, 3 for panel mIF, and 1 for H & E. For each experiment, 2 serial sections of normal human tonsil tissue were taken for control experiments, 1 for the yang reference control experiment and 1 for the IgG isotype control, all specimens were obtained from domestic tissue chip service company.

b. To evaluate the Precision (Precision) of polychromatic immunofluorescence results, 2 samples from lung and pancreatic cancer each were examined, at least 10 serial sections each, wherein: 9 per sample were used for the reproducibility experiments and 1 slice was used for H & E staining. For each experiment, 2 serial sections of normal human tonsil tissue were taken for control experiments, 1 for the yang reference control experiment and 1 for the IgG isotype control, all specimens were obtained from domestic tissue chip service company.

(2) HE quality control: and judging the HE dyeing result of the sections according to the HE reading result fed back by the pathologist, wherein the HE dyeing result judging standard is shown in the following table.

Table 24HE quality control Standard

* And (3) injection: all five parameters need to be within the reference range to be determined as the samples of the level, and if one parameter does not match, the samples of the next level are determined. Risk samples are typically not taken, but may be tried on-line, but the end result may be worse than a qualified sample.

(3) Accuracy analysis:

a. at least 3 samples were tested to assess accuracy.

b. The single IHC results serve as references for mIF.

c. In the analysis of the results, IHC or mIF staining was scanned using a Vectra polar scanner. Full scan and digital imaging analysis will be performed on 10 samples. The scanned image of the mIF was analyzed by imaging HALO software (or equivalent software) and then the cell density and proportion of each antibody staining of antibodies (CD 3, CD4, CD8, OX40, FOXP3, panCK) in a single mIF was compared to the cell density and proportion of a single IHC.

d. The staining results for each slide will be shown as positive cell count per square millimeter for each marker and as percentage of positive cells in total cells. The results of the IHC or stain same label are ranked. (1) Loading an image of a single IHC to the HALO; (2) the density of positive cells stained with each antibody was calculated and formulated as: positive cell count/image area. (3) In IHC and mIF staining results, the spearman correlation coefficient Rs for positive cell density for each antibody was calculated.

e. The correlation of individual IHC and mIF is detected using the Szelman correlation coefficient Rs. The strength of correlation is described using Rs: 0.20-0.39, "weak"; 0.40-0.59, "medium"; 0.60-1.0, "strong". Accuracy studies can only be validated when the Rs value is 0.6 or more.

(4) Precision analysis:

a. for accurate assessment, FFPE samples of 3 tumors will be subjected to a repeat experiment in at least three batches, with 3 sections per patient per experiment for antibody panel detection. And each experiment required 1 slice for the yang reference control detection and 1 slice for the isotype control detection.

b. Three batches to evaluate the accuracy of the assay should be performed by at least two different personnel for at least two days to cover as many variables as possible.

c. The stained sections were scanned by Vectra polar or equivalent methods. The 9 slices in each sample will be subjected to full slice scanning and digital imaging analysis. The HALO or equivalent software analyzes the images and then compares the staining results of one batch with the staining results of other batches. (1) An image of the mIF of one sample of each batch is loaded to the HALO. (2) The density and percentage of positive cells stained for each antibody was formulated: positive cell count/image area and percentage of positive cells in total cells. (3) The c.v. value for each antibody staining was calculated.

d. The positive cell density and ratio (positive cell count/image area percentage) of each antibody were calculated to give c.v. values. Only when c.v. is less than or equal to 0.2, the precision assessment will be considered as passing; if C.V >0.2, we will maintain the machine stable, control laboratory environmental conditions, ensure more stable experimental conditions, repeat the experiment again, and then calculate c.v. values using the data of the repeated experiment; the slice scan results are excluded from the calculation because the slice cannot yield a valid result for any reason. In addition, the evaluation of the batch including the sample is repeated, and the c.v. value is recalculated in the repeated experimental results.

(5) Verification result:

a. the sample meets the quality control requirement: the pathologist interpreted the H & E staining results of the samples as shown in the following table.

Table 25 sample H & E quality control results

b. Dyeing result: in lung and pancreatic cancer samples meeting quality control requirements, automated staining was performed with Bond RX using Vectra Pol aris panoramic scanning. The staining results revealed that subcellular localization was correct (cell membrane localization included CD3, CD4, CD8, OX40, cytoplasmic localization panCK, nuclear localization FOXP 3). Analysis of staining accuracy: 3 tumor FFPE samples, 3 sections each were taken for mIF detection and 6 sections for IHC detection. Using HALO we performed analytical processing on the images, as well as statistical analysis (fig. 21, 22). According to the data source: positive cell density and positive cell proportion of IHC and mIF assays for lung and pancreatic cancer samples.

c. Correlation analysis: the correlation of IHC and mIF is calculated using the Szelman correlation coefficient R. Intensity of the correlation is described using R: 0.20-0.39, "weak"; 0.40-0.59, "medium"; 0.60-1.0, "strong". As shown in fig. 21 and 22, fig. 21 shows the results of positive cell density correlation analysis of IHC and mIF detection in lung cancer and pancreatic cancer samples, and fig. 22 shows the results of positive cell proportion correlation analysis of IHC and mIF detection in lung cancer and pancreatic cancer samples. It can be seen that the expression of 6 proteins of CD3, CD4, CD8, OX40, FOXP3 and panCK was detected using IHC and mIHC, and the value of the Szechwan correlation coefficient R was 0.6 or more, thus passing the accuracy verification.

d. Precision analysis: FFPE samples of 2 tumors each of lung cancer and pancreatic cancer were subjected to repeated experiments in 3 batches, with 3 sections taken for each patient for detection per experiment. And each experiment required 1 slice for the yang reference control detection and 1 slice for the isotype control detection. Using HALO, we performed analytical processing on the images, as well as statistical analysis, the results are shown in fig. 23 and the table below. FIG. 23 is the staining results (200X) of repeated experiments in lung and pancreatic cancer samples. Rows 1 through 9 are replicate slides 1 through 9, respectively; columns 1-8 are panorama, DAPI, FOXP3, OX40, CD8, CD3, panCK, and CD4, respectively.

e. The c.v. value of positive cell density (positive cell count/percent image area) was calculated for each antibody and the average value was calculated (table 26). When the positive cell Density (Overall Density) is greater than 100/mm ² When C.V. is less than or equal to 0.2, the precision evaluation is carried out; when the positive cell density is less than 100/mm ² C.V. is less than or equal to 0.4 by precision evaluation. Since the c.v. satisfies the corresponding conditions, the precision verification was passed.

Table 26 results of verification of precision of mIF detection in lung cancer and pancreatic cancer samples

Verification conclusion: sample H by pathologist&E, judging a dyeing result, wherein a sample meets the quality control requirement; staining results revealed that subcellular localization was correct (cell membrane localization included CD3, CD4, CD8, OX40, cytoplasmic localization) Position panCK, nuclear localization FOXP 3); the accuracy verification result shows that the density of positive cells and the proportion correlation coefficient are both larger than 0.6, and the acceptance criterion is met. The results of the precision verification show that when the positive cell Density (Overall Density) is greater than 100/mm ² The c.v. average distribution is about 0.139 to 0.194; when the positive cell density is less than 100/mm ² The c.v. average distribution is about 0.297 to 0.299, meeting acceptance criteria. The performance verification is proved to be accepted, and can be used for detecting lung cancer and pancreatic cancer samples.

In one embodiment, the full-automatic multicolor immunofluorescence detection kit and method for lung cancer and pancreatic cancer samples provided by the invention realize automatic detection, and multiple immunofluorescence experiments are carried out on a single slice, so that the expression condition of six antigen targets can be presented simultaneously, and the utilization rate of the single tissue samples, especially the precious samples, is greatly improved.

In one embodiment, the sample is saved for the patient, so that the sample has enough quantity to be used as other detection items.

In one embodiment, the automated staining can perform high-throughput testing, standardized operation is realized, 30 pathological sections can be detected simultaneously at most in a single time, the defects of long manual operation time and large error are avoided, and a simple and efficient detection method is provided for clinical pathologists.

In one embodiment, the kit achieves automated detection of standardized operations, which have been validated for performance, with high accuracy and precision.

In one embodiment, the kit and the detection method reduce the requirements of the traditional detection method on the tissue sample, the puncture sample can also meet the requirements, and the problem of poor dyeing repeatability of the micro tissue sample is avoided.

The foregoing description of the invention has been presented for purposes of illustration and description, and is not intended to be limiting. Several simple deductions, modifications or substitutions may also be made by a person skilled in the art to which the invention pertains, based on the idea of the invention.

Claims

1. A method for staining a lung cancer and/or pancreatic cancer sample, comprising

OX40、CD3、FOXP3、CD4、CD8、panCK。

2. the method according to claim 1, wherein the number of kinds of the staining agent used in the staining step corresponds to the number of kinds of the antibody;

optionally, in the staining step, the primary antibody corresponding to the antigen and the staining agent are paired and stained in sequence as follows:

anti-OX 40 monoclonal antibody paired Opal 690, anti-CD 3 monoclonal antibody paired Opal 620, anti-FOXP 3 monoclonal antibody paired Opal570, anti-CD 4 monoclonal antibody paired Opal 520, anti-CD 8 monoclonal antibody paired Opal 480, anti-panCK monoclonal antibody paired Opal 780.

3. The staining method according to claim 1, wherein the staining step comprises repeating antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, staining, and antibody removal in the order of the antigens;

optionally, in the staining step, the secondary antibody used in the secondary antibody incubation is labeled with horseradish peroxidase.

4. The staining method according to claim 1, wherein in the staining step, after the first 5 antigens are stained, the sample is blocked by using a blocking solution, and then the 6 th antigen is sequentially subjected to primary antibody incubation, secondary antibody incubation, signal amplification staining, antibody removal, staining by using a corresponding stain, and staining by using a nuclear stain, so as to obtain a stained sample;

optionally, the stain used in signal amplification staining comprises Opal TSA-DIG dye;

optionally, the nuclear stain comprises DAPI.

5. The method of staining according to claim 1, wherein in the staining step, the sample comprises a cancer tissue section.

6. The method of staining according to claim 1, further comprising an imaging step comprising performing continuous spectral imaging of the stained sample to obtain a corresponding stained image;

optionally, the method further comprises a detection step of detecting the dyed image to obtain a detection result.

7. A sample or image obtained by the staining method according to any one of claims 1 to 6.

8. An image analysis method, comprising: biomarker analysis calculations are performed on the fluorescence expression data of the target region of the image of claim 7 to obtain a biomarker value for the sample.

9. A kit for detecting a lung and/or pancreatic cancer sample, comprising the following antibodies:

10. The kit of claim 9, further comprising a staining agent, wherein the number of staining agents corresponds to the number of antibody species;

optionally, the stain comprises a fluorescent stain;

optionally, the staining agent comprises:

Opal 690、Opal 620、Opal 570、Opal 520、Opal 480、Opal 780；

alternatively, the pairing relationship of the antibody and the staining agent is as follows:

anti-OX 40 monoclonal antibody paired Opal 690, anti-CD 3 monoclonal antibody paired Opal 620, anti-FOXP 3 monoclonal antibody paired Opal570, anti-CD 4 monoclonal antibody paired Opal 520, anti-CD 8 monoclonal antibody paired Opal 480, anti-panCK monoclonal antibody paired Opal 780;

optionally, the fluorescent dye Opal TSA-DIG is also included;

optionally, the kit further comprises at least one of an antigen retrieval liquid, a horseradish peroxidase-labeled secondary antibody, a blocking liquid, and an anti-fluorescence quencher.