CN115047192A

CN115047192A - Multiple fluorescence immunohistochemical analysis kit for non-small cell lung cancer and use method thereof

Info

Publication number: CN115047192A
Application number: CN202210699788.4A
Authority: CN
Inventors: 张媛祯
Original assignee: Suzhou Baidao Medical Technology Co ltd
Current assignee: Suzhou Baidao Medical Technology Co ltd
Priority date: 2022-06-20
Filing date: 2022-06-20
Publication date: 2022-09-13

Abstract

The invention relates to a multiple fluorescence immunohistochemical analysis kit for non-small cell lung cancer, which comprises: endogenous peroxidase blocking agent, monoclonal antibody group, antigen repairing buffer solution, HRP enzyme-labeled immune color reagent and tyramine fluorescent staining solution kit; the monoclonal antibody group comprises a CD8 monoclonal antibody, a PD-L1 monoclonal antibody and a CK-PAN monoclonal antibody; the tyramine fluorescent staining solution kit comprises TSA fluorescent staining solutions with at least three different excitation wavelengths. The invention utilizes the TSA technology to carry out high-density in-situ labeling on the target protein, improves the detection sensitivity and greatly improves the signal-to-noise ratio. TSA signal is not affected by microwave method, and can be retained after antibody removal, and TSA is covalently bonded and is very stable. The next round of staining detection can be carried out after the MWT microwave method treatment, and the interference of the antibody in the previous round of staining can not be caused.

Description

Multiple fluorescence immunohistochemical analysis kit for non-small cell lung cancer and use method thereof

Technical Field

The invention relates to the field of tumor diagnosis, in particular to a multiple fluorescence immunohistochemical analysis kit for non-small cell lung cancer and a using method thereof.

Background

Recent cancer burden data indicate that the incidence and mortality of lung cancer is very high. Non-small cell lung cancer (NSCLC) accounts for approximately 85% of all lung cancers. The majority of lung cancer patients are non-small cell lung cancers. Non-minicells are a disease caused by smoking, prolonged exposure to chemicals, contamination of the atmosphere, or chronic infection of the lungs. Surgery, chemotherapy (platinum-based treatment regimens), radiation therapy, combination radiotherapy and targeted therapy (alone or in combination) are still the current therapies for lung cancer. First, surgical removal of the tumor and nearby lymph nodes is the most consistent and effective treatment for early stage disease. Later, when surgery is not an ideal solution, radiation and/or chemotherapy may be recommended. In recent years, with the progress of advanced treatment regimens, a great number of immunotherapy regimens and epigenetic active drugs have been leaped.

Over the past decade, more and more solid tumor therapies have introduced T cell-targeting immunomodulators to block the immune checkpoints CTLA-4(Ipilimumab) and PD-1 or PDL-1 and achieve significant efficacy. At present, 8 immune checkpoint inhibitors are approved to be on the market in China and are used for treating various cancer types. In tumor immunotherapy represented by PD-1/L1 inhibitors, positive expression of PD-L1 is closely related to treatment remission, but the predicted effect is relatively limited. To improve efficacy prediction and to screen for a more benefical patient population, researchers have proposed increasing CD8 based on PD-L1 and analyzing the association between high density CD8+ and PD-L1+ cells and improved survival of non-small cell lung cancer patients under Durvalumab (Devalumab) treatment. However, the detection means and reagents of the prior art cannot predict the effectiveness of the immune checkpoint inhibitors with high sensitivity and high specificity, and limit the prediction capability of the curative effect of the immune checkpoint inhibitors.

Disclosure of Invention

Technical problem to be solved

In view of the above disadvantages and shortcomings of the prior art, the present invention provides a multiple fluorescence immunohistochemical assay kit for non-small cell lung cancer, which can effectively predict the effectiveness of immune checkpoint inhibitors and has extremely high atopy and sensitivity. The invention also relates to a method for using the assay kit.

(II) technical scheme

In order to achieve the purpose, the invention adopts the main technical scheme that:

in a first aspect, the present invention provides a multiple fluorescent immunohistochemical assay kit for non-small cell lung cancer, comprising: endogenous peroxidase blocking agent, monoclonal antibody group, antigen repairing buffer solution, HRP enzyme-labeled immunochromatography reagent and tyramine fluorescent staining solution kit; the monoclonal antibody group comprises a CD8 monoclonal antibody, a PD-L1 monoclonal antibody and a CK-PAN monoclonal antibody; the tyramine fluorescent staining solution kit comprises TSA fluorescent staining solutions with at least three different excitation wavelengths.

According to the preferred embodiment of the invention, the tyramine fluorescent staining solution kit comprises TSA488, TSA555, TSA640 staining solutions and a signal amplification agent.

According to a preferred embodiment of the invention, the kit further comprises (ready-to-use) an anti-fluorescence quenching encapsulated tablet.

Preferably, the above reagents can be selected according to the product specification according to the amount of the detection piece, and the adding amount is generally 100 muL/piece when carrying out single-piece detection; the monoclonal antibody group is a ready-to-use reagent.

In a second aspect, the present invention provides a method for using the above multiple fluorescence immunohistochemical assay kit for non-small cell lung cancer, which comprises the steps of dewaxing and hydrating paraffin sections of tissues, and then performing continuous three times of antibody incubation, immuno-visualization, fluorescent staining and signal amplification; in particular, the amount of the solvent to be used,

respectively adopting a CD8 monoclonal antibody, a PD-L1 monoclonal antibody and a CK-PAN monoclonal antibody to perform co-incubation with tissues repaired by an antigen repair liquid microwave method and treated by an endogenous peroxidase blocking agent, using an HRP enzyme-labeled immunochromatographic reagent to perform HRP enzyme-labeled immunochromatographic after each incubation, then correspondingly using one TSA fluorescent staining solution in the tyramine fluorescent staining solution kit to perform staining treatment, and then performing fluorescent detection; wherein, a TSA fluorescent staining solution is correspondingly used after each antibody is incubated, and different antibodies do not share the same staining solution;

by the treatment, the number of CD8 single positive, PD-L1 single positive, CK-PAN single positive, total under-lens cells, CD8+ PD-L1 double positive and CK-PAN + PD-L1 double positive cells in the sample are detected.

According to the preferred embodiment of the present invention, the method for performing antigen retrieval using the antigen retrieval solution comprises: pouring the antigen retrieval liquid into a retrieval cup, heating the antigen retrieval liquid for 3min by a microwave oven at 700-800W high fire, placing the dewaxed glass slide into the retrieval cup, continuing to heat the glass slide for 15min by a microwave oven at 100-150W low fire, and cooling the glass slide to room temperature. Before the step is carried out, a preliminary experiment is suggested to be carried out without placing a sample, and whether the liquid level is higher than the sample after 15min is detected, so that dry pieces are avoided. The antigen retrieval solution cannot be reused.

According to the preferred embodiment of the invention, after the PD-L1 monoclonal antibody and the tissue which is repaired by the antigen repairing solution and inactivated by the endogenous peroxidase are co-incubated, an HRP enzyme-labeled immune color development reagent is used for immune color development, and TSA555 staining solution and a signal amplification agent in a tyramine fluorescent staining solution kit are used for fluorescent staining and signal amplification treatment;

co-incubating a CD8 monoclonal antibody and a tissue which is repaired by an antigen repairing solution and inactivated by endogenous peroxidase, performing immunochromatography by using an HRP enzyme-labeled immunochromatography reagent, and performing fluorescent staining and signal amplification treatment by using a TSA488 staining solution and a signal amplification agent in a tyramine fluorescent staining solution kit;

and (3) co-incubating the CK-PAN monoclonal antibody and the tissue which is repaired by the antigen repairing solution and inactivated by endogenous peroxidase, performing immunochromatography by using an HRP enzyme-labeled immunochromatography reagent, and performing fluorescent staining and signal amplification treatment by using a TSA640 staining solution and a signal amplification agent in a tyramine fluorescent staining solution kit.

In the use of the kit of the present invention, the antibody incubation order, the color development order, the color of the fluorescent dye corresponding to the antibody (the type of the staining solution), and the like can be interchanged at will, and the sequence and the correspondence are not limited to the above.

According to the preferred embodiment of the present invention, after the three consecutive antibody incubations, the immuno-color development and the fluorescent staining and the signal amplification are completed, the mounting preservation is performed. The mounting method comprises the following steps: dripping an anti-fluorescence quenching blocking tablet, sealing the blocking tablet by using a cover glass to avoid air bubbles, and sealing the edge of the cover glass by using transparent nail polish for long-term storage.

In a third aspect, the invention also provides application of the multiple fluorescence immunohistochemical analysis kit for the non-small cell lung cancer in predicting/evaluating the effectiveness of the immune checkpoint inhibitor on the treatment of the non-small cell lung cancer.

Preferably, the prediction/evaluation method comprises:

(1) calculating the number of single positive CD8, single positive PD-L1, single positive CK-PAN and total cells under the lens in the detection sample by using analysis software;

(2) calculating the number of CD8+ PD-L1 double-positive and CK-PAN + PD-L1 double-positive cells in the detection sample by using analysis software;

(3) when the double positive of CD8+ PD-L1 is more than 2.14, the curative effect of the immune checkpoint inhibitor on the patient from which the sample to be detected is obtained is judged to be high, and otherwise, the curative effect is low.

(III) advantageous effects

The invention has the beneficial effects that: the kit is mainly based on a multiple immunofluorescence technology, and the principle is that a TSA technology (fully named as Tvramide neutral America) utilizes horseradish peroxidase (HRP) to carry out high-density in-situ labeling on target protein, so that the detection sensitivity is improved, and the signal to noise ratio is greatly improved. TSA signal is not affected by microwave method, and can be retained after antibody removal, and TSA is covalently bonded and is very stable. After the MWT microwave method treatment, the next round of staining detection can be performed without worrying about interference from the antibody in the previous round of staining. Therefore, multicolor labeling can be achieved even if a primary antibody derived from the same species is used (TSA binding mode is stable, the last staining result is not lost after microwave repair, and color cross with the last target staining result due to insufficient repair elution even if the next antibody species is the same). Paraffin section samples can be stained multiple times by using different fluorescence labeled TSA for each round of staining.

Drawings

FIG. 1 shows the expression of PD-L1 (TSA 555 as a staining solution) in a sample detected by the assay kit provided by the present invention.

FIG. 2 shows the expression of CD8 (the staining solution is TSA488) in a sample detected by the assay kit provided by the present invention.

FIG. 3 shows the expression of CK-PAN (the staining solution is TSA640) in a sample detected by the assay kit of the present invention.

FIG. 4 shows the co-expression of PD-L1+ CD8+ CK-PAN, which is obtained by superimposing expressions of PD-L1 (staining solution TSA555), CD8 (staining solution TSA488) and CK-PAN (staining solution TSA640) by using analysis software.

FIG. 5 is a diagram showing the PD-L1+ CD8+ CK-PAN co-expression profile obtained by abstracting the PD-L1+ CD8+ CK-PAN co-expression shown in FIG. 4 by using analysis software.

Detailed Description

For the purpose of better explaining the present invention and to facilitate understanding, the present invention will be described in detail by way of specific embodiments with reference to the accompanying drawings.

The invention provides a multiple fluorescence immunohistochemical analysis kit for non-small cell lung cancer, which comprises: endogenous peroxidase blocking agent, monoclonal antibody group, antigen repairing buffer solution, HRP enzyme-labeled immunochromatography reagent and tyramine fluorescent staining solution kit; the monoclonal antibody group comprises a CD8 monoclonal antibody, a PD-L1 monoclonal antibody and a CK-PAN monoclonal antibody; the tyramine fluorescent staining solution kit comprises TSA fluorescent staining solutions with at least three different excitation wavelengths.

Preferably, the tyramine fluorescent staining solution kit comprises TSA488, TSA555 and TSA640 staining solutions and a signal amplification agent.

Preferably, the kit further comprises (ready-to-use) an anti-fluorescence quenching encapsulated tablet.

The following is a method of using the multiple fluorescent immunohistochemical assay kit of the preferred embodiment of the present invention, the method comprising:

1. sample preparation: and (3) slicing the paraffin, baking the slices in a constant temperature oven at 60 ℃ for 2 hours, and storing the slices at room temperature for later use.

2. Dewaxing and hydrating:

a. the paraffin sections were soaked in fresh xylene for 15 min. The treatment was performed 2 times.

b. The paraffin sections were sequentially placed in gradient alcohol (100%, 95%, 85%, 70%) for 3min each time.

c. Washing with distilled water for 1 time, and PBS for 2 times, each for 3 min.

3. Antigen retrieval: pouring the 1x antigen retrieval liquid into the retrieval cup, and heating for 3min by a microwave oven with high fire of 700-. And (3) placing the dewaxed glass slide in a repair cup, continuing to heat for 15min under low fire of 100 DEG and 150W, and then cooling to room temperature. (note: before this step, it is recommended to perform a preliminary experiment without sample, detect whether the liquid level is higher than the sample after 15min, avoid dry sheet, and the antigen retrieval fluid cannot be reused.)

4. Inactivation of endogenous peroxidase: removing the excessive liquid, enclosing the tissue with an immunohistochemical pen, adding 1-3 drops of instant endogenous peroxidase blocker to completely cover the tissue, incubating at room temperature for 15min, and washing with PBS for 2 times, each time for 3 min.

PD-L1 monoclonal antibody incubation: 1-3 drops of ready-to-use PD-L1 monoclonal antibody were added to completely cover the tissue, incubated in a 37 ℃ wet box for 60min, and washed 3 times with PBS for 2min each.

6. And (3) secondary antibody incubation: adding 1-3 drops of instant HRP enzyme-labeled immunochromatography reagent, completely covering the tissue, incubating in a wet box at 37 ℃ for 30min, and washing with PBS for 3 times, 2min each time.

7. Fluorescent staining and signal amplification: and removing the washing liquid on the slide, adding 0.2mL of signal amplification liquid and 1 mu L of 200X TSA555 staining solution into the preparation tube, uniformly mixing, dropwise adding the prepared TSA555 working solution, and immersing the sample region. Incubate at room temperature for 10min, wash 3 times with PBS, 2min each time.

As shown in FIG. 1, the result of fluorescence staining for detecting positive expression of PD-L1 in a sample by using the assay kit provided by the present invention is shown.

8. Antigen retrieval: pouring the 1x antigen retrieval liquid into a retrieval cup, and heating for 3min by a microwave oven with high fire (700-. And (3) placing the dewaxed glass slide in a repair cup, continuing to heat for 15min by low fire (100- & 150W), and then cooling to room temperature. (note: it is recommended to perform a preliminary experiment without placing a sample before this step, and to check whether the liquid level is higher than the sample after 15min, to avoid dry tablets, the antigen retrieval solution cannot be reused.)

9. Inactivation of endogenous peroxidase: removing the excessive liquid, using an immunohistochemical pen to enclose the tissue, adding 1-3 ready-to-use drops of endogenous peroxidase blocker to completely cover the tissue, incubating at room temperature for 15min, and washing with PBS for 2 times, each time for 3 min.

CD8 monoclonal antibody incubation: 1-3 drops of ready-to-use CD8 monoclonal antibody were added to completely cover the tissue, incubated in a wet box at 37 ℃ for 60min, and washed 3 times with PBS for 2min each.

11. And (3) secondary antibody incubation: adding 1-3 drops of instant HRP enzyme-labeled immunochromatographic reagent to completely cover the tissue, incubating in a wet box at 37 ℃ for 30min, and washing with PBS for 3 times, 2min each time.

12. Fluorescent staining and signal amplification: the washing solution on the slide was removed, 0.2mL of signal amplification solution and 1. mu.L of 200 XTSA 488 staining solution were added to the preparation tube, and after mixing, the prepared TSA488 working solution was added dropwise to immerse the sample region. Incubate at room temperature for 10min, wash 3 times with PBS, 2min each time.

As shown in FIG. 2, the fluorescence staining result of the positive expression of CD8 in the sample detected by the assay kit provided by the present invention is shown.

13. Antigen retrieval: pouring the 1x antigen retrieval liquid into a retrieval cup, and heating for 3min by a microwave oven with high fire (700-. And (3) placing the dewaxed glass slide in a repair cup, continuing to heat for 15min by low fire (100- & 150W), and then cooling to room temperature. (note: it is recommended to perform a preliminary experiment without placing a sample before this step, and to check whether the liquid level is higher than the sample after 15min, to avoid dry tablets, the antigen retrieval solution cannot be reused.)

14. Inactivation of endogenous peroxidase: removing the excessive liquid, enclosing the tissue with an immunohistochemical pen, adding 1-3 drops of instant endogenous peroxidase blocker to completely cover the tissue, incubating at room temperature for 15min, and washing with PBS for 2 times, each time for 3 min.

CK-PAN monoclonal antibody incubation: adding 1-3 drops of ready-to-use CK-PAN monoclonal antibody to completely cover the tissue, incubating at 37 ℃ for 60min in a wet box, and washing 3 times with PBS for 2min each time.

16. And (3) secondary antibody incubation: adding 1-3 drops of instant HRP enzyme-labeled immunochromatographic reagent to completely cover the tissue, incubating in a wet box at 37 ℃ for 30min, and washing with PBS for 3 times, 2min each time.

17. Fluorescent staining and signal amplification: the washing solution on the slide was removed, 0.2mL of signal amplification solution and 1. mu.L of 200 XTSA 640 staining solution were added to the preparation tube, and after mixing, the prepared TSA640 working solution was added dropwise to immerse the sample region. Incubate at room temperature for 10min, wash 3 times with PBS, 2min each time.

As shown in FIG. 3, the result of fluorescent staining for CK-PAN positive expression in a sample is shown by using the assay kit provided by the present invention.

18. Sealing: dripping anti-fluorescence quenching sealing tablets, sealing the sealing tablets by using a cover glass to avoid bubbles, and sealing the edges of the cover glass by using transparent nail polish for long-term storage.

The three fluorescent staining patterns of PD-L1 positive expression, CD8 positive expression and CK-PAN positive expression shown in FIG. 1, FIG. 2 and FIG. 3 were superimposed by computer software to obtain the co-expression fluorescent staining pattern of PD-L1+ CD8+ CK-PAN as shown in FIG. 4. FIG. 5 is a fluorescent staining profile of the co-expression of PD-L1+ CD8+ CK-PAN resulting from the abstraction process of FIG. 4.

Then, the analysis software is used for calculating the number of CD8 single positive, PD-L1 single positive, CK-PAN single positive, total cells under the lens, CD8+ PD-L1 double positive, CK-PAN + PD-L1 double positive cells and the like in the detection sample. When the double positive of CD8+ PD-L1 is more than 2.14, the curative effect of the immune checkpoint inhibitor on the patient from which the sample to be detected is obtained is judged to be high; conversely, it is indicative that the immune checkpoint inhibitor is less effective in treating the patient. The calculation method is as follows:

CD8+ single positive is the number of single positive cells of tumor infiltrating marginal tissue CD 8;

(ii) PD-L1+ single positive (number of tumor infiltrating marginal tissue PD-L1+ single positive cells);

(iii) CK-PAN + single positive (number of tumor infiltrating border tissue CK-PAN + single positive cells);

(iv) the number of cells which are double positive in CD8+ PD-L1+ tumor-associated region CD8+ PD-L1 +;

(CK-PAN + PD-L1+ double positive area CK-PAN + PD-L1+ double positive cell number)

Sixthly, the ratio of CD8+ (the number of CD8 single positive cells divided by the total number of cells of tumor infiltration border tissues) is multiplied by 100 percent;

(vii) PD-L1+ ratio (PD-L1 single positive cells/total tumor infiltrating border tissue cells) × 100%;

(v) ratio of CD8+ PD-L1+ (CD8+ PD-L1+ double positive cells/total cells of tumor infiltrating border tissue) × 100%;

ninthly, CK-PAN + accounts for (CK-PAN single positive cell number divided by total cell number of tumor infiltration border tissue) multiplied by 100%;

CD8, PD-L1 and CK-PAN expression, as shown by multiple immunofluorescence assays in FIGS. 4-5, were calculated using analytical software according to the methods described above and summarized in the following Table (Table 1):

TABLE 1

Note: tumor-associated regions scored refer to the regions occupied by tumor cells and connective tissue tumor stroma within and adjacent to their associated tumors.

Wherein the number of PD-L1+ single positive cells comprises both PD-L1 positive tumor cells (TC +) in the entire delineated tumor area and PD-L1 positive infiltrating immune cells (IC +) in the entire delineated tumor area.

Wherein PD-L1 and CD8+ double positively express tumors, showing significant activated antigen expression and enhanced T cell signaling.

The multiple fluorescence immunohistochemical analysis kit has strong specificity and high sensitivity for the effectiveness prediction of the immune checkpoint inhibitor, and provides conditions for realizing accurate medication. The following description will be given with reference to specific application examples.

Application example

120 non-small cell lung cancer biopsy samples are selected for research, multiple fluorescence immunohistochemistry is adopted to carry out CD8, PD-L1 and CK-PAN expression detection on the same sample, and analysis software is used for detecting the co-expression condition of CD8 and PD-L1. High expression of PD-L1 is defined as expression of > 25% of tumor cells at any staining intensity.

TABLE 2.120 survival of non-small cell Lung cancer patients

As can be seen from the data in the table, the combination of CD8+ and PD-L1+ correlates well with the prognosis of patients with non-small cell lung cancer treated with Durvalumab.

The reagent information of primary antibody, immunodominant reagent, TSA fluorescent dye, and anti-fluorescence quenching blocking tablet used in this application example are shown in the following table (Table 3):

TABLE 3 information on the reagents for anti-and immuno-chromogenic reagents and fluorescent dyes

Finally, it should be noted that: the above embodiments are only used to illustrate the technical solution of the present invention, and not to limit the same; while the invention has been described in detail and with reference to the foregoing embodiments, it will be understood by those skilled in the art that: the technical solutions described in the foregoing embodiments may still be modified, or some or all of the technical features may be equivalently replaced; and the modifications or the substitutions do not make the essence of the corresponding technical solutions depart from the scope of the technical solutions of the embodiments of the present invention.

Claims

1. A multiple fluorescence immunohistochemical assay kit for non-small cell lung cancer, comprising: endogenous peroxidase blocking agent, monoclonal antibody group, antigen repairing buffer solution, HRP enzyme-labeled immune color reagent and tyramine fluorescent staining solution kit; the monoclonal antibody group comprises a CD8 monoclonal antibody, a PD-L1 monoclonal antibody and a CK-PAN monoclonal antibody; the tyramine fluorescent staining solution kit comprises TSA fluorescent staining solutions with at least three different excitation wavelengths.

2. The kit of claim 1, wherein the tyramine fluorescent staining solution kit comprises TSA488, TSA555, TSA640 staining solution and a signal amplification agent.

3. The kit of claim 1, further comprising: an anti-fluorescence quenching encapsulated tablet.

4. The method of using the multiple fluorescent immunohistochemical assay kit for non-small cell lung cancer according to any one of claims 1 to 3, comprising:

after paraffin sections of tissues are dewaxed and hydrated, continuous three times of antibody incubation, immune development, fluorescent staining and signal amplification are carried out; in particular, the amount of the solvent to be used,

respectively adopting a CD8 monoclonal antibody, a PD-L1 monoclonal antibody and a CK-PAN monoclonal antibody to perform co-incubation with tissues repaired by an antigen repair liquid microwave method and treated by an endogenous peroxidase blocking agent, performing HRP enzyme-labeled immunochromatography by using an HRP enzyme-labeled immunochromatography reagent after each incubation, then correspondingly dyeing one of TSA fluorescent dyeing liquids in the tyramine fluorescent dyeing liquid kit, and then performing fluorescent detection; wherein, a TSA fluorescent staining solution is correspondingly used after each antibody is incubated, and different antibodies do not share the same staining solution;

by the above treatment, the number of cells which are single positive for CD8, single positive for PD-L1, single positive for CK-PAN, total number of cells under the lens, double positive for CD8+ PD-L1 and double positive for CK-PAN + PD-L1 in the sample were detected.

5. The use of the composition according to claim 4, wherein the antigen retrieval method using the antigen retrieval solution comprises: pouring the antigen repairing liquid into a repairing cup, heating for 3min by a microwave oven with high temperature of 700 plus 800W, placing the dewaxed glass slide into the repairing cup, continuing to heat for 15min by low temperature of 100 plus 150W, and cooling to room temperature.

6. The use method of claim 4, wherein the PD-L1 monoclonal antibody is co-incubated with the tissue repaired by the antigen repair solution and inactivated by endogenous peroxidase, then the tissue is subjected to immunochromatography by using an HRP enzyme-labeled immunochromatography reagent, and fluorescence staining and signal amplification are performed by using TSA555 staining solution and a signal amplification agent in a tyramine fluorescence staining solution kit;

7. The use method of claim 4, wherein the antibody incubation, the immuno-visualization, the fluorescent staining and the signal amplification are performed three times in succession before mounting and preservation; the sealing method comprises the following steps: and (4) dropwise adding an anti-fluorescence quenching blocking piece, and blocking the piece by using a cover glass to avoid bubbles.

8. Use of the multiple fluorescent immunohistochemical assay kit for non-small cell lung cancer of any one of claims 1-3 for predicting/assessing the effectiveness of an immune checkpoint inhibitor for treatment of non-small cell lung cancer.

9. The use according to claim 8, wherein the prediction/evaluation method comprises:

(1) calculating the number of CD8 single positive, PD-L1 single positive, CK-PAN single positive and total cells under the lens in the detection sample by using analysis software;

(3) when the double positive of CD8+ PD-L1 is more than 2.14, the effectiveness of the immune checkpoint inhibitor on the treatment of the patient from which the sample to be detected is obtained is judged to be high, and otherwise, the effectiveness is low.