Brightfield multicolor immunohistochemical staining method and kit

Abstract

The present invention provides a bright field multicolor immunohistochemical staining method, which can simultaneously detect multiple antigens on a tissue section under bright field color development conditions, firstly labeling the first antigen with the first first antibody and the second antibody labeled with horseradish peroxidase, then using a color developer for color development, recording the tissue image after color development, and then using a decolorizer for decolorization, repeating the above steps, and sequentially completing the detection of multiple antigens, as well as a bright field multicolor immunohistochemical staining kit. The present invention can provide a multi-channel visual molecular pathology detection method with simpler equipment requirements and lower detection costs for research fields such as tumors and their immune microenvironments, tissue damage and healing, etc.

Description

Technical Field

[0001] The present invention relates to the technical field of immunohistochemistry, and more specifically to a bright field multicolor immunohistochemical staining method and a kit. Background Art

[0002] Immunohistochemistry technology is based on the principle of specific binding between antigen and antibody. It uses antibodies linked to enzymes or fluorescein to label target antigens in situ in tissues, and then detects the colored products generated by the enzyme-substrate reaction or the fluorescent signals of fluorescein to locate and quantitatively detect the target antigens.

[0003] Multicolor immunohistochemistry is a technique for simultaneously labeling multiple antigens on a tissue section. This technique can intuitively display the co-localization and co-expression of multiple antigens in tissue cells, and plays an irreplaceable and important role in the research fields of tumors and their immune microenvironment, tissue damage and healing. Existing multicolor immunohistochemistry techniques are all based on fluorescence imaging. Usually, multiple antibodies linked to different fluorescein are used to label multiple antigens in the tissue simultaneously or sequentially, so that 3 to 5 antigens can be detected simultaneously on a tissue section. There is also a technique that first labels and records the fluorescence signal of the first antibody, then extracts and quenches its signal, and then labels the second antibody, repeats this cycle, and uses image processing software to superimpose the images of each antigen, so that 3 to 8 antigens can be detected simultaneously on a tissue section. This type of technology requires the use of fluorescence microscopy equipment and fluorescein-linked antibodies, which have high requirements for instruments and equipment, and the market price of reagents is high.

[0004] Brightfield immunohistochemistry is a technique that uses enzyme-linked antibodies to label target antigens, then adds substrates to react with the enzymes to produce colored products, and then observes and analyzes the colored products under brightfield conditions to detect target antigens. This technique only requires brightfield microscopy equipment to observe the results, and enzyme-linked antibodies are widely used in clinical and scientific research fields and have low market prices. However, the products of the enzyme-substrate color development system have a large molecular weight and will cover adjacent antigens. Existing brightfield immunohistochemistry techniques can only detect 1 to 3 antigens at the same time, and cannot detect more antigens on a tissue section at the same time. Summary of the invention

[0005] In view of the problems existing in the prior art, the purpose of the present invention is to provide a bright-field multicolor immunohistochemical staining method and a kit, which can realize the simultaneous detection of 4 to 10 antigens on a tissue section under bright-field color development conditions.

[0006] To achieve the above-mentioned purpose of the invention, the present invention provides a bright field multicolor immunohistochemical staining method, which can detect multiple antigens on a tissue section at the same time under bright field color development conditions, firstly labeling the first antigen with the first first antibody and the second antibody labeled with horseradish peroxidase, then using a color developer to develop the color, recording the tissue image after the development, and then using a decolorizer to decolorize, repeating the above steps, and completing the detection of multiple antigens in sequence; wherein,

[0007] The color developer is composed of liquid A and liquid B; liquid A is an N-dimethylformamide solution containing 2 to 5 m / v% 3,3',5,5'-tetramethylbenzidine; liquid B is an aqueous solution containing 25 to 30 v / v% ethanol, 2.5 to 5 v / v% methanol, 1 to 2 m / v% sodium citrate, 0.3 to 0.5 m / v% sodium tungstate and 0.2 to 0.5 v / v% phosphoric acid; when used, liquid A and liquid B are prepared and used in a volume ratio of 1:40 to 1:80;

[0008] The decolorizing agent is an aqueous solution containing 1 to 2 m / v% sodium dodecyl sulfate, 0.5 to 1 m / v% sodium chloride, 1 to 2 v / v% ethylene glycol, 1 to 2 m / v% glycine and 0.1 to 0.3 m / v% ammonium chloride, and having a pH value of 3.0 to 3.5.

[0009] Preferably, the bright field multicolor immunohistochemical staining method comprises the following steps:

[0010] Step 1: Tissue sections are baked, dewaxed and rehydrated;

[0011] Step 2: Staining of cell nuclei on tissue sections;

[0012] Step 3: Record the nuclear staining image of the tissue section using a bright field microscope;

[0013] Step 4: antigen retrieval of tissue sections, followed by washing with phosphate buffered saline;

[0014] Step 5: labeling the first antigen in the tissue section with the first primary antibody and washing with phosphate buffer;

[0015] Step 6: label the first antibody with the second antibody labeled with horseradish peroxidase, and wash with phosphate buffer;

[0016] Step 7, developing the tissue sections with the developer, stopping the development with the developer solution B, washing with distilled water, and sealing with distilled water;

[0017] Step 8: Using a bright field microscope to record the image of the tissue section stained with a chromogen;

[0018] Step 9, soaking the tissue sections in the decolorizing agent until they are completely decolorized;

[0019] Step 10: Wash the tissue sections with distilled water and then with phosphate buffer;

[0020] Step 11, repeating steps 5 to 10 until the Nth antigen staining is completed, where N is greater than or equal to 4;

[0021] Step 12: Use image processing software to give pseudo-color to the images of nuclear staining and developer staining of tissue sections, superimpose the pseudo-color images, and generate bright-field multi-color immunohistochemistry detection result images.

[0022] Preferably, in the bright-field multicolor immunohistochemical staining method, N is any integer from 4 to 10.

[0023] The present invention also provides a bright field multicolor immunohistochemical staining kit, comprising a developer and a decolorizer; wherein:

[0024] The color developer is composed of liquid A and liquid B; liquid A is an N-dimethylformamide solution containing 2 to 5 m / v% 3,3',5,5'-tetramethylbenzidine; liquid B is an aqueous solution containing 25 to 30 v / v% ethanol, 2.5 to 5 v / v% methanol, 1 to 2 m / v% sodium citrate, 0.3 to 0.5 m / v% sodium tungstate and 0.2 to 0.5 v / v% phosphoric acid; when used, liquid A and liquid B are prepared and used in a volume ratio of 1:40 to 1:80;

[0025] The decolorizing agent is an aqueous solution containing 1 to 2 m / v% sodium dodecyl sulfate, 0.5 to 1 m / v% sodium chloride, 1 to 2 v / v% ethylene glycol, 1 to 2 m / v% glycine and 0.1 to 0.3 m / v% ammonium chloride, and having a pH value of 3.0 to 3.5.

[0026] Preferably, the bright field multicolor immunohistochemical staining kit further comprises an antigen repair solution, a phosphate buffer, a plurality of first antibodies, a plurality of horseradish peroxidase-labeled second antibodies matching the first antibodies, a hematoxylin staining solution and a differentiation solution.

[0027] Preferably, in the bright field multicolor immunohistochemical staining kit, the multiple first antibodies are 4 to 10 first antibodies.

[0028] Compared with the prior art, the present invention has the following beneficial effects:

[0029] (1) Under bright field conditions, 4 to 10 antigens can be detected simultaneously on a tissue section.

[0030] (2) In the bright-field multicolor immunohistochemical staining step of the present invention, except for the developer and decolorizer, other consumables, reagents, and software (including but not limited to glass slides, cover slips, antigen retrieval solution, phosphate buffer, multiple primary antibodies, horseradish peroxidase-labeled secondary antibodies matching the primary antibodies, hematoxylin staining solution and differentiation solution, and image processing software) are not particularly limited and various commercially available products can be freely selected.

[0031] (3) The present invention can provide a multi-channel visual molecular pathology detection method with simpler equipment requirements and lower detection costs for research fields such as tumors and their immune microenvironment, tissue damage and healing. BRIEF DESCRIPTION OF THE DRAWINGS

[0032] Figure 1 This is a picture of the results of single-color and multi-color immunohistochemical staining of human tonsil tissue;

[0033] Figure 2 This is the result of bright-field multi-color immunohistochemical staining of human tonsil tissue and the pseudo-color image of positive signals;

[0034] Figure 3 This is a pseudo-color overlay of bright-field-based multi-color immunohistochemical staining of human tonsil tissue;

[0035] Figure 4 This is a picture of the results of single-color and multi-color immunohistochemical staining of human appendix tissue;

[0036] Figure 5 This is the bright-field based multi-color immunohistochemical staining result of human appendix tissue and the pseudo-color image of positive signals;

[0037] Figure 6 This is a pseudo-color overlay of bright-field-based multi-color immunohistochemical staining of human appendix tissue;

[0038] Figure 7 It is the result of multi-color immunohistochemical staining of human thyroid cancer tissue based on bright field and pseudo-color image of positive signals;

[0039] Figure 8 This is a pseudo-color overlay of bright-field multi-color immunohistochemical staining of human thyroid cancer tissue. DETAILED DESCRIPTION

[0040] The present invention is further described in detail below in conjunction with embodiments and drawings so that those skilled in the art can implement the invention with reference to the description.

[0041] It should be understood that terms such as “having”, “including” and “comprising” used herein do not exclude the existence or addition of one or more other elements or combinations thereof.

[0042] It should be noted that the experimental methods described in the following embodiments are conventional methods unless otherwise specified, and the reagents and materials can be obtained from commercial channels unless otherwise specified.

[0043] Example 1: Preparation of colorants and decolorants for bright field multicolor immunohistochemical staining

[0044] (1) Preparation of color developer A solution

[0045] Take a clean glass beaker with a volume of 10mL, weigh 0.2g 3,3',5,5'-tetramethylbenzidine, and add it to the beaker; then draw 5mL N,N-dimethylformamide, add it to the beaker, and stir with a glass rod until it is completely dissolved. Transfer the liquid into a brown glass reagent bottle, seal it and store it at 4℃ for later use.

[0046] (2) Preparation of color developer solution B

[0047] Take a clean glass beaker with a volume of 200mL, add 50mL of distilled water; weigh 1.5g of anhydrous sodium citrate and 0.45g of sodium tungstate dihydrate, add them to the beaker in turn, and stir with a glass stirring rod until completely dissolved; take 0.6mL of phosphoric acid, 4mL of methanol, and 26mL of anhydrous ethanol, respectively, add them to the beaker in turn, and stir evenly with a glass stirring rod; add distilled water to a total volume of 100mL, and stir evenly again. Transfer the liquid into a clean brown HDPE plastic reagent bottle with a volume of 120mL, seal it and store it at 4℃ for use.

[0048] (3) Preparation of color developer

[0049] After the color developer A solution and the color developer B solution are placed at room temperature to return to temperature, the color developer A solution and the color developer B solution are mixed at a volume ratio of 1:50 to prepare a color developer working solution, which is prepared and used immediately.

[0050] (4) Preparation of decolorizing agent

[0051] Take a clean glass beaker with a volume of 500mL, add 100mL of distilled water; weigh 3g of sodium dodecyl sulfate, 1.5g of sodium chloride, 3g of glycine and 0.4g of ammonium chloride, add them to the beaker in turn, and stir with a glass stirring rod until completely dissolved; draw 3mL of ethylene glycol, add it to the beaker and stir evenly; add distilled water to a total volume of 200mL, and stir evenly again. Adjust the pH value of the liquid to 3.0 with hydrochloric acid. Transfer the liquid into a clean brown HDPE plastic reagent bottle with a volume of 250mL, seal it and store it at 4℃ for later use.

[0052] Example 2: Bright field multicolor immunohistochemical staining of human tonsil tissue

[0053] Materials and Methods

[0054] Paraffin-embedded human tonsil tissue was prepared into 4 μm thick continuous paraffin sections using conventional paraffin sectioning technology. The sections were picked up with immunohistochemistry slides (Shitai, 188105, China), baked on a 65°C baking table for 2 hours, dewaxed by immersion in xylene for 20 minutes, and rehydrated with 100%, 95%, 80%, 70% gradient ethanol and tap water.

[0055] One slice was taken for HE (hematoxylin and eosin) staining. A hematoxylin and eosin staining kit (Solarbio, G1120, China) was used. The specific steps were carried out according to the instructions of the kit. A bright field pathology whole slide scanner (Jiangfeng, KF-PRO, China) was used to scan and record the staining results.

[0056] Ten sections were taken for conventional immunohistochemical single-color staining. The reagents used were purchased from ZSGB-Bio (China), including: phosphate buffer (ZLI-9061), antigen retrieval solution (ZLI-9069), primary antibodies CD20 (ZA-0293), CD68 (ZM-0060), CKpan (ZM-0069), S100 (ZM-0224), CD34 (ZA-0550), CD8 (ZA-0508), CD3 (ZA-0503), SMA (ZM-0003), Vimentin (ZM-0260), Ki67 (ZM-0167), anti-rabbit / mouse horseradish peroxidase-labeled secondary antibody (PV-6000), 3,3-diaminobenzidine (3,3'-Diaminobenzidine, DAB) color development solution (ZLI-9017). The nucleus was counterstained using a hematoxylin-eosin staining kit (Solarbio, G1120, China). The specific steps were performed according to the kit instructions. A bright field pathology whole slide scanner (Jiangfeng, KF-PRO, China) was used to scan and record the staining results.

[0057] Take one slice and perform immunohistochemical multicolor staining. The color developer and decolorizer are prepared by the method described in Example 1. The remaining reagents are the same as those used for conventional immunohistochemical single-color staining. The specific steps are as follows:

[0058] (1) After conventional baking, dewaxing and rehydration, the tissue sections were immersed in phosphate buffer at room temperature for 10 minutes;

[0059] (2) Add hematoxylin staining solution (Solarbio, G1120, China) to the slices to completely cover the tissue, let it stand at room temperature for 5 minutes, wash with tap water, add differentiation solution (Solarbio, G1120, China) to completely cover the tissue, let it stand at room temperature for 3 seconds, wash with tap water for 15 minutes until the tissue turns blue, and seal the slices with distilled water;

[0060] (3) Use a bright-field pathology whole-slide scanner to scan and record tissue images after hematoxylin staining;

[0061] (4) Perform conventional high-pressure repair of tissue sections with antigen repair solution for 2.5 minutes;

[0062] (5) Soak the sections in phosphate buffer at room temperature for 10 minutes;

[0063] (6) The staining order of the ten indicators is CD20, CD68, CKpan, S100, CD34, CD8, CD3, SMA, Vimentin, and Ki67 (the above staining order is only an application example in this embodiment and cannot be understood as a limitation on the staining order. The immunohistochemical multicolor staining results are not affected by the indicator staining order);

[0064] (7) Add the first antibody working solution of the first indicator to the slice and incubate in a 37°C wet box for 1 hour;

[0065] (8) The sections were washed with phosphate buffer, and the working solution of the secondary antibody labeled with horseradish peroxidase was added dropwise, and incubated in a wet box at 37°C for 30 min;

[0066] (9) Wash the sections with phosphate buffer, add freshly prepared color developer working solution to completely cover the tissue, incubate in a 37°C wet box for 7 minutes, immerse the sections in color developer solution B to terminate the color development reaction, then wash the sections with distilled water and seal the sections with distilled water;

[0067] (10) Use a bright-field pathology whole-slice scanner to scan and record the tissue images stained with chromogens;

[0068] (11) Soak the sections in a decolorizer at 37°C for 40 minutes until completely decolorized;

[0069] (12) The sections were washed with distilled water and soaked in phosphate buffer at room temperature for 10 min.

[0070] (13) Repeat steps (7) to (12) until 10 primary antibodies are labeled one by one.

[0071] (14) Photoshop image processing software (Adobe, USA) was used to give pseudocolor to the hematoxylin and chromogen staining result images, and the pseudocolor images were superimposed to generate multicolor immunohistochemical detection result images.

[0072] result

[0073] See Figure 1 , Figure 2 and Figure 3 .

[0074] Figure 1In the figure, HE (hematoxylin and eosin) staining and images of 10 indicators in part A are derived from 11 tissue sections, respectively, and Hematoxylin (hematoxylin) staining and images of 10 indicators in part B are derived from the same tissue section. Part A is the test result of using conventional immunohistochemistry single-color staining method, with each section marked with one indicator, using 3,3-diaminobenzene for color development, and positive signals appear brown. Part B is the multicolor immunohistochemistry test result of using the staining method described in the present invention, with 10 indicators marked on one section at the same time, using the color developer described in the present invention for color development, and positive signals appear blue-green. Figure 1 It can be seen that a total of 10 indicators, CD20, CD68, CKpan, S100, CD34, CD8, CD3, SMA, Vim, and Ki67, are all positively expressed to varying degrees in human tonsil tissue. The positive signals of the above 10 indicators are consistent in location and quantification in human tonsil tissue by using conventional bright field single color (Part A) and bright field multicolor immunohistochemical staining (Part B) of the present invention. Figure 1 The magnification ratio under the microscope is 5 times, scale bar = 2mm.

[0075] Figure 2 In the figure, all images are from the same tissue area of ​​the same tissue section. Part A is the nucleus hematoxylin staining; and the bright field image of 10 indicators of multicolor immunohistochemical staining using the method of the present invention, and the positive signal is blue-green in the bright field. Part B is the pseudo-color image formed after processing by extracting the positive signal in the bright field image using image processing software, and the specific color of the pseudo-color can be freely set. Figure 2 It can be seen that, after software processing, the blue-green positive signal in the bright field multicolor immunohistochemical staining result of the present invention can be processed to form a pseudo-color image, and the specific color of the pseudo-color can be freely set to facilitate the distinction and observation of the detection results. Figure 2 The magnification under the microscope is 400 times, scale bar = 100 μm.

[0076] Figure 3 In the figure, the left and right images are from the same tissue area of ​​the same tissue section. The left side shows the superposition of the cell nucleus (Nu) and 6 indicators including Vim, S100, CKpan, Ki67, and SMA. The right side shows the superposition of the cell nucleus (Nu) and 6 indicators including CD20, CD3, CD8, CD68, and CD34. Figure 3 The magnification under the microscope is 400 times, scale bar = 100 μm.

[0077] Depend on Figure 2 and Figure 3It can be seen that, after software processing, the blue-green positive signal in the bright field multicolor immunohistochemical staining result of the present invention can be processed to form a pseudo-color image, and the specific color of the pseudo-color can be freely set to facilitate the distinction and observation of the detection results.

[0078] Example 3: Bright field multicolor immunohistochemical staining of human appendix tissue

[0079] Materials and Methods

[0080] Paraffin-embedded human appendix tissue was prepared into 4 μm thick continuous paraffin sections using conventional paraffin sectioning technology. The sections were picked up with immunohistochemistry slides (Matsunami, PRO-01, Japan), baked on a 65°C baking table for 2 hours, dewaxed by immersion in xylene for 20 minutes, and rehydrated with 100%, 95%, 80%, 70% gradient ethanol and tap water.

[0081] One slice was taken and HE staining was performed. The specific steps were the same as those described in Example 2.

[0082] Ten sections were taken for conventional immunohistochemical single-color staining. The reagents used were purchased from MXB-Bio (China), including: phosphate buffer (PBS-0060), antigen retrieval solution (MVS-0099), primary antibodies CK20 (MAB-0834), Desmin (MAB-0766), S100 (RAB-0150), Vimentin (MAB-0735), Ki67 (MAB-0672), CD16 (MAB-0869), CD4 (RMA-0620), CD20 (Kit-0001), CD31 (MAB-0720), CD14 (MAB-0512), anti-rabbit / mouse horseradish peroxidase-labeled secondary antibody (containing DAB) (KIT-5020). Hematoxylin-eosin staining kit (Solarbio, G1120, China) was used for counterstaining of cell nuclei. The specific steps were carried out according to the instructions of the kit. A bright-field pathology whole-slide scanner was used to scan and record the staining results.

[0083] Take 1 slice and perform immunohistochemical polychromatic staining. The developer and decolorant are prepared by the method described in Example 1. The remaining reagents are the same as the reagents used for conventional immunohistochemical monochromatic staining. The specific steps are the same as those described in Example 2. The staining order of the ten indicators is CK20, Desmin, S100, Vimentin, Ki67, CD16, CD4, CD20, CD31, CD14 (the above staining order is only an application example in this embodiment and cannot be understood as limiting the staining order. The immunohistochemical polychromatic staining results are not affected by the indicator staining order).

[0084] result

[0085] See Figure 4 , Figure 5 and Figure 6 .

[0086] Figure 4 In the figure, HE (hematoxylin and eosin) staining and images of 10 indicators in part A are derived from 11 tissue sections, respectively, and Hematoxylin (hematoxylin) staining and images of 10 indicators in part B are derived from the same tissue section. Part A is the test result of using conventional immunohistochemistry single-color staining method, with each section marked with one indicator, using 3,3-diaminobenzene for color development, and positive signals appear brown. Part B is the multicolor immunohistochemistry test result of using the staining method described in the present invention, with 10 indicators marked on one section at the same time, using the color developer described in the present invention for color development, and positive signals appear blue-green. Figure 4 It can be seen that CK20, Desmin, S100, Vimentin, Ki67, CD16, CD4, CD20, CD31, CD14, a total of 10 indicators, are all positively expressed in human appendix tissue to varying degrees. The positive signals of the above 10 indicators are consistent in location and quantity in human appendix tissue by using conventional bright field single color (Part A) and bright field multicolor immunohistochemical staining (Part B) of the present invention. Figure 4 The magnification ratio under the microscope is 2 times, scale bar = 5mm.

[0087] Figure 5 In the figure, all images are from the same tissue area of ​​the same tissue section. Part A is a nucleus hematoxylin staining; and a bright field image of 10 indicators of multicolor immunohistochemical staining using the method of the present invention, where the positive signal appears blue-green in the bright field. Part B is a pseudo-color image formed after processing by extracting the positive signal in the bright field image using image processing software, and the specific color of the pseudo-color can be freely set. Figure 5 The magnification under the microscope is 400 times, scale bar = 100 μm.

[0088] Figure 6In the figure, all images are from the same tissue area of ​​the same tissue section. A is a pseudo-color image of the cell nucleus (Nu) and 10 indicators including CK20, Desmin, S100, Vimentin, Ki67, CD16, CD4, CD20, CD31, and CD14. B is a pseudo-color overlay of the cell nucleus (Nu) and the above 10 indicators. C is a pseudo-color overlay of the cell nucleus (Nu) and CK20, S100, CD16, CD4, and CD31, which is a total of 6 indicators. D is a pseudo-color overlay of the cell nucleus (Nu) and CK20, Desmin, Veminten, Ki67, CD4, and CD14, which is a total of 6 indicators, which is reset to the pseudo-color. E is a pseudo-color overlay of the cell nucleus (Nu) and CK20, CD31, Ki67, S100, CD4, and Desmin, which is a total of 6 indicators, which is reset to the pseudo-color. Figure 6 The magnification under the microscope is 400 times, scale bar = 100 μm.

[0089] Depend on Figure 5 and Figure 6 It can be seen that, after software processing, the blue-green positive signal in the bright field multicolor immunohistochemical staining result of the present invention can be processed to form a pseudo-color image, and the specific color of the pseudo-color can be freely set to facilitate the distinction and observation of the detection results.

[0090] Example 4: Bright field multicolor immunohistochemical staining of human thyroid cancer tissue

[0091] Materials and Methods

[0092] Paraffin-embedded tissues of human thyroid cancer were prepared into 4-μm-thick continuous paraffin sections using conventional paraffin sectioning techniques. The sections were scooped out using immunohistochemistry slides (Matsunami, PRO-01, Japan), baked on a 65°C baking table for 2 hours, dewaxed by immersion in xylene for 20 minutes, and rehydrated with 100%, 95%, 80%, 70% gradient ethanol and tap water.

[0093] One section was taken for immunohistochemical multicolor staining. The color developer and decolorizer were prepared by the method described in Example 1. The reagents used included: phosphate buffer (Solarbio, P1010, China), antigen retrieval solution (Solarbio, C1038, China), primary antibody diluent (Solarbio, A1810, China), anti-rabbit / mouse horseradish peroxidase-labeled secondary antibody (RecordBio, RCB054, China). The primary antibodies were concentrated and diluted with primary antibody diluent according to the following volume ratios before use: Tg (1:200, ABclonal, A3407, China), LAG3 (1:1000, Abcam, ab209236, USA), TIM3 (1:500, Abcam, ab246852, USA), PD1 (1:50, Abcam, ab52587, USA), CD56 (1:200, ABclonal, A7913, China) ), CD16 (1:1000, ABclonal, A23541, China), Ki67 (1:5000, 27309-1-AP, Proteintech, China), CD20 (1:1000, 24828-1-AP, Proteintech, China), CD8 (1:100, MA5-14548, Thermo, USA), CD68 (1:200, 14-0688-82, Thermo, USA). Hematoxylin-eosin staining kit (Solarbio, G1120, China) was used for counterstaining of cell nuclei. The specific steps were the same as those described in Example 2. The staining order of the ten indicators is Tg, LAG3, TIM3, PD1, CD56, CD16, Ki67, CD20, CD8, and CD68 (the above staining order is only an application example in this embodiment and cannot be understood as a restriction on the staining order. The immunohistochemical multicolor staining results are not affected by the indicator staining order).

[0094] Figure 7 In the figure, all images are from the same tissue area of ​​the same tissue section. Part A is the nucleus hematoxylin staining; and the bright field image of 10 indicators of multicolor immunohistochemical staining using the method of the present invention, and the positive signal is blue-green in the bright field. Part B is the pseudo-color image formed after processing by extracting the blue-green positive signal in the bright field image using image processing software, and the specific color of the pseudo-color can be freely set. Figure 7 It can be seen that, after software processing, the blue-green positive signal in the bright field multicolor immunohistochemical staining result of the present invention can be processed to form a pseudo-color image, and the specific color of the pseudo-color can be freely set to facilitate the distinction and observation of the detection results. Figure 7 The magnification under the microscope is 400 times, scale bar = 100 μm.

[0095] Figure 8 In the figure, all images are from the same tissue area of ​​the same tissue section. A is a pseudo-color image of the cell nucleus (Nu) and the 10 indicators of Tg, LAG3, TIM3, PD1, CD56, CD16, Ki67, CD20, CD8, and CD68. B is a pseudo-color overlay of the cell nucleus (Nu) and the above 10 indicators. C is a pseudo-color overlay of the cell nucleus (Nu) and Tg, CD56, LAG3, PD1, and CD20, which are 6 indicators in total. D is a pseudo-color overlay of the cell nucleus (Nu) and CD56, PD1, Ki67, TIM3, and LAG3, which are 6 indicators in total. E is a pseudo-color overlay of the cell nucleus (Nu) and CD16, Ki67, CD56, CD8, and CD68, which are 6 indicators in total. Figure 8 The magnification under the microscope is 400 times, scale bar = 100 μm.

[0096] Depend on Figure 7 and Figure 8 It can be seen that, after software processing, the blue-green positive signal in the bright field multicolor immunohistochemical staining result of the present invention can be processed to form a pseudo-color image, and the specific color of the pseudo-color can be freely set to facilitate the distinction and observation of the detection results.

[0097] The results of the above examples prove that the present invention can detect 10 antigens on the same tissue section under bright field conditions. For one of the indicators, the immunohistochemical staining results of the present invention and the conventional method are consistent in positioning and quantification. The bright field multicolor immunohistochemical staining method and kit of the present invention are in line with expectations and are technically feasible.

[0098] Although the embodiments of the present invention have been disclosed as above, they are not limited to the applications listed in the specification and the implementation modes, and they can be fully applied to various fields suitable for the present invention. For those familiar with the art, additional modifications can be easily implemented. Therefore, without departing from the general concept defined by the claims and the scope of equivalents, the present invention is not limited to the specific details and the illustrations shown and described herein.

Claims

1. A bright field multicolor immunohistochemical staining method, characterized in that: Under bright field color development conditions, multiple antigens are detected on a tissue section. The first antigen is first labeled with the first primary antibody and the second antibody labeled with horseradish peroxidase, and then a color developer is used for color development. The tissue image after color development is recorded, and then a decolorizer is used for decolorization. The above steps are repeated to complete the detection of multiple antigens in sequence; wherein, The color developer is composed of liquid A and liquid B; liquid A is an N-dimethylformamide solution containing 2 to 5 m / v% 3,3',5,5'-tetramethylbenzidine; liquid B is an aqueous solution containing 25 to 30 v / v% ethanol, 2.5 to 5 v / v% methanol, 1 to 2 m / v% sodium citrate, 0.3 to 0.5 m / v% sodium tungstate and 0.2 to 0.5 v / v% phosphoric acid; when used, liquid A and liquid B are prepared and used at a volume ratio of 1:40 to 1:80; the tissue section is colored with the color developer, and the color development is stopped with the liquid B of the color developer; The decolorizing agent is an aqueous solution containing 1 to 2 m / v% sodium dodecyl sulfate, 0.5 to 1 m / v% sodium chloride, 1 to 2 v / v% ethylene glycol, 1 to 2 m / v% glycine and 0.1 to 0.3 m / v% ammonium chloride, and having a pH value of 3.0 to 3.

5.

2. The bright field multicolor immunohistochemical staining method according to claim 1, characterized in that: The steps include: Step 1: Tissue sections are baked, dewaxed and rehydrated; Step 2: Staining of cell nuclei on tissue sections; Step 3: Record the nuclear staining image of the tissue section using a bright field microscope; Step 4: antigen retrieval of tissue sections, followed by washing with phosphate buffered saline; Step 5: labeling the first antigen in the tissue section with the first primary antibody and washing with phosphate buffer; Step 6: label the first antibody with the second antibody labeled with horseradish peroxidase, and wash with phosphate buffer; Step 7, developing the tissue sections with the developer, stopping the development with the developer solution B, washing with distilled water, and sealing with distilled water; Step 8: Using a bright field microscope to record the image of the tissue section stained with a chromogen; Step 9, soaking the tissue sections in the decolorizing agent until they are completely decolorized; Step 10: Wash the tissue sections with distilled water and then with phosphate buffer; Step 11, repeating steps 5 to 10 until the Nth antigen staining is completed, where N is greater than or equal to 4; Step 12: Use image processing software to give pseudo-color to the images of nuclear staining and developer staining of tissue sections, superimpose the pseudo-color images, and generate bright-field multi-color immunohistochemistry detection result images.

3. The bright field multicolor immunohistochemical staining method according to claim 2, characterized in that: The N is any integer from 4 to 10.

4. Bright field multicolor immunohistochemical staining kit, characterized in that: It includes a color developer, a decolorizer, an antigen repair solution, a phosphate buffer, multiple first antibodies, multiple horseradish peroxidase-labeled second antibodies matching the first antibodies, a hematoxylin staining solution, and a differentiation solution; wherein, The color developer is composed of liquid A and liquid B; liquid A is an N-dimethylformamide solution containing 2 to 5 m / v% 3,3',5,5'-tetramethylbenzidine; liquid B is an aqueous solution containing 25 to 30 v / v% ethanol, 2.5 to 5 v / v% methanol, 1 to 2 m / v% sodium citrate, 0.3 to 0.5 m / v% sodium tungstate and 0.2 to 0.5 v / v% phosphoric acid; when used, liquid A and liquid B are prepared and used at a volume ratio of 1:40 to 1:80; the tissue section is colored with the color developer, and the color development is stopped with the liquid B of the color developer; The decolorizing agent is an aqueous solution containing 1 to 2 m / v% sodium dodecyl sulfate, 0.5 to 1 m / v% sodium chloride, 1 to 2 v / v% ethylene glycol, 1 to 2 m / v% glycine and 0.1 to 0.3 m / v% ammonium chloride, and having a pH value of 3.0 to 3.

5.

5. The bright field multicolor immunohistochemical staining kit according to claim 4, characterized in that: The plurality of first antibodies is 4 to 10 first antibodies.

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