Immunohistochemical multiplex sequential staining with cross-talk blocking agent and application thereof

Abstract

The application relates to a color blocking agent for immunohistochemical multiplex sequential staining and application thereof, and belongs to the biomedical technology field. The color blocking agent is mainly composed of a buffer, an oxidizing agent and an eluent. The color blocking agent is used for immunohistochemical multiplex sequential staining, can effectively prevent cross staining, is suitable for a full-automatic immunohistochemical machine, is mild in condition and does not cause damage to tissues, and can be widely applied to full-automatic multiplex sequential staining in immunohistochemical pathological examination.

Description

Technical Field

[0001] This invention relates to a color-blocking agent for multiple sequential staining in immunohistochemistry and its application, belonging to the field of biomedical technology. Background Technology

[0002] Immunohistochemical pathology utilizes the principle of specific antigen-antibody binding. First, a mouse anti-human primary antibody (selected according to the specific situation) specifically binds to the target antigen in the tissue. Then, a primary antibody amplification agent (rabbit anti-mouse antibody) binds to the primary antibody, amplifying the effect. Next, an HRP- or AP-labeled goat anti-rabbit secondary antibody binds to the amplification agent, and finally, a suitable chromogenic agent is used for color development. Through antigen-antibody reactions and colorimetric reactions, the chemical components in cells or tissues are revealed, allowing for the in-situ determination of the distribution and content of certain chemical components within cells or tissues.

[0003] Currently, the disadvantages of conventional single-stain immunohistochemistry (IHC) are: ① A large number of stained sections are required for a single sample, necessitating the interpretation of staining results from multiple sections and comprehensive analysis to obtain a pathological diagnosis; ② IHC has strict requirements on sample size, but with technological advancements, patient tumor tissue samples are now obtained through small biopsies or fine-needle aspiration, yielding paraffin blocks with only a limited tumor content. From these limited portions, the pathology laboratory must prepare the necessary HE slides (usually two) for diagnosing the main subtype, as well as other slides supporting the diagnosis, thus presenting significant drawbacks.

[0004] To address this phenomenon, multiplex sequential staining technology was developed. Its principle involves sequentially performing immunohistochemistry using HRP and AP systems. The improvements of this technology are: ① it can display two colors on the same tissue section, labeling at least two antigen targets; ② it has no restrictions on the species of the primary antibody required; and ③ it reduces the amount of tissue used and the workload for physicians interpreting slides. However, this technology still has a critical problem: when detecting multiple antigen targets on the same slide, the staining of the previous antibody can be affected by the staining process of the next antibody, causing color mixing of the first antibody stain, which easily leads to misinterpretation of the results.

[0005] Cross-staining in multiple sequential staining can be caused by: ① binding between the primary antibody in the first staining step and the amplifying agent following the primary antibody in the second staining step, resulting in cross-staining; ② binding between the amplifying agent following the primary antibody in the first staining step and the primary antibody in the second staining step, resulting in cross-staining; ③ binding between the labeled secondary antibody in the first staining step and the amplifying agent following the primary antibody in the second staining step, resulting in cross-staining. Currently, conventional methods, such as blocking treatment with serum, mouse IgG, and rabbit IgG, cannot effectively solve the cross-staining problem. Summary of the Invention

[0006] To address the aforementioned problems, the first objective of this invention is to provide a cross-staining inhibitor for multiple sequential staining in immunohistochemistry, which effectively prevents cross-staining, operates under mild conditions, and does not damage tissues.

[0007] A second objective of this invention is to provide the application of this color-blocking agent in immunohistochemical multiple sequential staining.

[0008] To achieve the above objectives, the technical solution adopted by the present invention is as follows:

[0009] An immunohistochemical multiple serosa staining cross-linking blocking agent mainly comprises an oxidant, an eluent, and a buffer; the oxidant is one or more of hydrogen peroxide, sodium percarbonate, and nickel peroxide; the eluent is one or more of sodium dodecyl sulfate (SDS), deoxycholate ammonium, sodium cholate, sarcosine, Triton-X100, CHAPS, urea, dodecyl maltodextrin, Tween-20, Tween-80, and digitalis saponins; the mass ratio of the oxidant to the eluent is 0.1–5:0.1–5.

[0010] More preferably, the mass ratio of the oxidant to the eluent is 0.3-4:0.5-3.

[0011] The cross-color blocking agent provided by this invention can effectively prevent cross-color mixing between different colors during multiple sequential staining-re-staining processes in immunohistochemistry, obtaining accurate staining results and effectively improving work efficiency and the accuracy of immunohistochemical pathological diagnosis. In this cross-color blocking agent, the oxidant inactivates the primary antibody, post-primary antibody amplification agent, and labeled secondary antibody that retain binding ability in the first staining procedure. Then, the unbound primary antibody, post-primary antibody amplification agent, and labeled secondary antibody are thoroughly eluted by the elution agent, thereby preventing cross-color mixing.

[0012] Preferably, the pH of the buffer is 6.5 to 10.0.

[0013] There are no special requirements for the choice of buffer. Commonly used buffers include tris(hydroxymethyl)aminomethane (Tris), phosphate (PBS), N-2-hydroxyethylpiperazine-N-3-propanesulfonic acid (HEPPS), tris(hydroxymethyl)methyl-3-aminopropanesulfonic acid (TAPSN), 3-(N-morphine)propanesulfonic acid (MOPS), diethanolamine (DEA), and triethanolamine (TRA).

[0014] More preferably, the pH of the buffer is 7.0 to 8.5.

[0015] More preferably, the buffer has a pH of 7.5.

[0016] This pH range is sufficient to maintain the pH environment required for the already bound antigen and antibody.

[0017] Preferably, it further includes one, two, or three of the following: stabilizer, defoamer, and preservative; the mass ratio of the oxidant to the stabilizer is 0.1–5:0.1–20; the mass ratio of the oxidant to the defoamer is 0.1–5:0.1–5; and the mass ratio of the oxidant to the preservative is 0.1–5:0.01–1.

[0018] More preferably, the mass ratio of the oxidant to the stabilizer is 0.3-4:1.2-17; the mass ratio of the oxidant to the defoamer is 0.3-4:0.2-4; and the mass ratio of the oxidant to the preservative is 0.2-0.4:0.05-0.8.

[0019] In the color-crossing inhibitor of this invention, the stabilizer maintains the chemical balance of the inhibitor, reduces surface tension, and stabilizes the reagent; the preservative delays spoilage caused by microorganisms or chemical changes. The combined action of the stabilizer and preservative facilitates the preservation of the color-crossing inhibitor and extends its shelf life. The defoamer primarily prevents the formation of bubbles during heating when using an automated immunohistochemistry machine, thus avoiding uneven staining.

[0020] More preferably, the stabilizer is one or more of bovine serum albumin, calf serum, glycine, glycerol, and polyethylene glycol 6000-20000; the defoamer is polydimethylsiloxane; and the preservative is any one of Proclin 950, gentamicin sulfate, and Kathon.

[0021] The stabilizers mentioned are conventional choices in reagents and can be selected and combined according to the storage time of the reagents and the type of eluent. Proclin 950, gentamicin sulfate and Kathon all have broad-spectrum antibacterial properties, are chemically stable and have very low toxicity, reducing the risk of manual operation.

[0022] Preferably, the oxidant has a mass content of 0.1% to 5% in the color-blocking agent.

[0023] More preferably, the oxidant has a mass content of 0.3-4% in the color-blocking agent.

[0024] More preferably, the amount of buffer used is 5-600 mM.

[0025] The application of cross-staining agents in immunohistochemical multiple sequential staining involves incubating with a cross-staining agent after the previous staining to block the cross-staining process before proceeding to the next sequential staining.

[0026] When using the color-crossing blocking agent of the present invention for multiple sequential staining in immunohistochemistry, the localization staining of different antibodies will not interfere with each other, effectively solving the problem of color cross-coloring, greatly improving the staining effect of multiple sequential staining technology, and making the results more accurate.

[0027] Preferably, the incubation blocking temperature is 40-60℃ and the time is 10 minutes.

[0028] This colorimetric inhibitor has mild blocking conditions, does not require heating above 80°C, and incubation at 40-60°C will not damage tissue, causing changes in tissue morphology and thus affecting the interpretation of disease results; at the same time, its blocking time is short, only about 10 minutes, which greatly saves time and improves staining efficiency.

[0029] Preferably, the immunohistochemical multiple sequential staining is performed manually or by a fully automated immunohistochemical multiple sequential staining method. Attached Figure Description

[0030] Figure 1 This is a diagram showing the effect of multiple sequential staining of lymphoma tissue using the color blocking agent prepared in Example 1 in Example 4 of the present invention.

[0031] Figure 2 This is a diagram showing the effect of multiple sequential staining on lymphoma tissue using the color blocking agent prepared in Example 2 in Example 5 of the present invention.

[0032] Figure 3 This is a diagram showing the effect of multiple sequential staining on lymphoma tissue using the color-blocking agent prepared in Example 3 in Example 6 of the present invention.

[0033] Figure 4 This is a diagram showing the effect of multiple sequential staining of lymphoma tissue using the color blocking agent prepared in Comparative Example 1 in Comparative Example 2 of the present invention.

[0034] Figure 5 This is a diagram showing the effect of multiple sequential staining on lymphoma tissue without the use of a cross-color blocking agent in Comparative Example 3 of the present invention;

[0035] Figure 6 This is a diagram showing the effect of multiple sequential staining of lung squamous cell carcinoma tissue using the color-blocking agent prepared in Example 1 in Example 7 of the present invention.

[0036] Figure 7 This is a diagram showing the effect of multiple sequential staining of lung squamous cell carcinoma tissue using the color-blocking agent prepared in Example 1 in Example 8 of the present invention.

[0037] Figure 8 This is a diagram showing the effect of multiple sequential staining of lung squamous cell carcinoma tissue using the color-blocking agent prepared in Example 1 in Example 9 of the present invention.

[0038] Figure 9This is a diagram showing the effect of multiple sequential staining of lung squamous cell carcinoma tissue using the color-blocking agent prepared in Comparative Example 1 in Comparative Example 4 of the present invention.

[0039] Figure 10 This is a diagram showing the effect of multiple sequential staining on lung squamous cell carcinoma tissue without the use of a cross-color blocking agent in Comparative Example 5 of the present invention. Detailed Implementation

[0040] The present invention will be further described in detail below with reference to specific embodiments. Unless otherwise specified, the equipment and reagents used in the embodiments, experimental examples and comparative examples are all commercially available.

[0041] The following is a brief introduction to some of the biological materials, experimental reagents, and experimental equipment involved in the following examples and experimental cases:

[0042] Reagent Information

[0043] Antigen retrieval buffer II, Henan Sainuo Biotechnology Co., Ltd., Product No.: SD6004; Immunochromatographic reagent (brown staining), Henan Sainuo Biotechnology Co., Ltd., Product No.: SD5300; Immunochromatographic reagent (red staining), Henan Sainuo Biotechnology Co., Ltd., Product No.: SD8013.

[0044] Experimental instruments

[0045] Fully automated immunohistochemical staining machine, Henan Sainote Biotechnology Co., Ltd., Model: CNT330.

[0046] I. Specific Embodiments of a Color Cross-Inhibiting Agent for Multiple Sequential Immunohistochemical Staining of the Present Invention

[0047] Example 1

[0048] This embodiment provides a cross-color blocking agent 1 for multiple sequential immunohistochemical staining, wherein the components are weighed or measured according to the table below:

[0049] Table 1 Formulation of Color Crosstalk Blocker 1

[0050]

[0051]

[0052] The cross-color blocking agent 1 for immunohistochemical multiple sequential staining provided in this embodiment is prepared as follows:

[0053] 1) According to the formula shown in Table 1, weigh out Tris, NaCl, PEG8000, sodium percarbonate, SDS and glycine in sequence, add them to purified water, mix well, adjust the pH to 7.5, and prepare reagent A;

[0054] 2) Add BSA, glycerol, Proclin 950 and polydimethylsiloxane to reagent A in sequence, and continue stirring to dissolve to obtain color-blocking agent 1.

[0055] Note: SDS is selected as the eluent in the formulation of this embodiment. Since SDS is a strong eluent, a variety of stabilizers are selected to provide certain protection for the bound antigen and antibody.

[0056] Example 2

[0057] This embodiment provides a cross-color blocking agent 2 for multiple sequential immunohistochemical staining, wherein the components are weighed or measured according to the table below:

[0058] Table 2 Formulation of Color Crosstalk Blocker 2

[0059] Components Added amount Tris 1.00g Nacl 4.00g BSA 2.00g PEG8000 5.00g Sodium percarbonate 10.00g SDS 15.00g glycerin 5.00ml glycine 2.00g Proclin950 0.50ml polydimethylsiloxane 10.00ml Purified water Adjust the volume to 1.00L.

[0060] The cross-color blocking agent 2 for multiple sequential immunohistochemical staining provided in this embodiment is prepared as follows:

[0061] 1) According to the formula shown in Table 2, weigh out Tris, NaCl, PEG8000, sodium percarbonate, SDS and glycine in sequence, add them to purified water, mix well, adjust the pH to 7.5, and prepare reagent B;

[0062] 2) Add BSA, glycerol, Proclin 950 and polydimethylsiloxane to reagent B in sequence, and continue stirring to dissolve to obtain color-blocking agent 2.

[0063] Example 3

[0064] This embodiment provides a cross-color blocking agent 3 for multiple sequential immunohistochemical staining, and the components are weighed or measured according to the table below:

[0065] Table 3 Formulation of Color Crosstalk Blocker 3

[0066] Components Added amount Tris 48.00g Nacl 72.00g BSA 27.00g PEG8000 50.00g Sodium percarbonate 40.00g SDS 30.00g glycerin 45.00ml glycine 40.00g Proclin950 8.00ml polydimethylsiloxane 42.00ml Purified water Adjust the volume to 1.00L.

[0067] The cross-color blocking agent 3 for multiple sequential immunohistochemical staining provided in this embodiment is prepared as follows:

[0068] 1) According to the formula shown in Table 3, weigh out Tris, NaCl, PEG8000, sodium percarbonate, SDS and glycine in sequence, add them to purified water, mix well, adjust the pH to 7.5, and prepare reagent C;

[0069] 2) Add BSA, glycerol, Proclin 950 and polydimethylsiloxane to reagent C in sequence, and continue stirring to dissolve to obtain color-blocking agent 3.

[0070] Comparative Example 1

[0071] The color-blocking agent formulation provided in this comparative example is shown in the table below. It does not contain an oxidant, and the remaining components are the same as those in Example 1.

[0072] Table 4 Formulation of the color-blocking agent in Comparative Example 1

[0073]

[0074]

[0075] The cross-color blocking agent 1 for immunohistochemical multiple sequential staining provided in this embodiment is prepared as follows:

[0076] 1) According to the formula shown in Table 1, weigh Tris, NaCl, PEG8000, SDS and glycine in sequence, add them to purified water, mix well, adjust the pH to 7.5, and prepare reagent A;

[0077] 2) Add BSA, glycerol, Proclin 950 and polydimethylsiloxane to reagent A in sequence, and continue stirring to dissolve to obtain Comparative Example 1.

[0078] II. Specific Examples of the Application of the Color Crossing Blocking Agent in Immunohistochemical Multiple Sequential Staining of the Present Invention

[0079] 2.1 Staining experiments in lymphoma tissue (see Examples 4-6 and Comparative Examples 2-3)

[0080] In the staining experiments on lymphoma tissue in Examples 4-6 and Comparative Examples 2-3, the tissue sections used were tissue sections from the same tissue block. As the tissue sections were prepared, the morphology of the tissue in the sections would change due to the irregularity of the tissue morphology, the large experimental volume, and the use of too many sections, which is a normal phenomenon.

[0081] Example 4

[0082] The application of the color-blocking agent in immunohistochemical multiple sequential staining in this embodiment involves using a fully automated immunohistochemical staining machine and the color-blocking agent of Embodiment 1 of this invention. In lymphoma tissue, Ki67 is first stained red, followed by CD3 stained brown, for multiple sequential staining. The specific implementation method is as follows:

[0083] 1. Dewaxing and hydration of paraffin tissue sections;

[0084] 2. Antigen retrieval: Antigen retrieval buffer II was used to perform high-pressure retrieval on the slides at 100℃ for 20 minutes, with a sample volume of 150ul.

[0085] 3. Add 3% to 3.5% peroxidase blocking agent to block endogenous peroxidase, incubate at room temperature for 5 minutes, and add 150 μL of sample.

[0086] 4. Incubation of the primary antibody in the first procedure: Incubate at 37°C for 30 minutes, with a sample volume of 150 μL.

[0087] 5. Incubation after primary antibody administration in the first procedure: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0088] 6. Incubation of the second antibody in the first procedure: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0089] 7. Rapid red color development: Incubate at room temperature for 8 minutes, and add 150ul of sample.

[0090] 8. Color-blocking agent: Block at 40-60℃ for 10 minutes, with a sample volume of 150ul.

[0091] 9. Incubation of the primary antibody in the second procedure: Incubate at 37°C for 30 minutes, with a sample volume of 150 μL.

[0092] 10. Incubation after primary antibody administration in the second procedure: Incubate at 37°C for 10 minutes, with a sample volume of 150 μL.

[0093] 11. Second procedure for secondary antibody incubation: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0094] 12. DAB color development: Incubate at room temperature for 5 minutes, and add 150ul of sample.

[0095] 13. Staining: Incubate with hematoxylin at room temperature for 1 minute, and add 150ul of sample.

[0096] 14. Seal the slide with water-based mounting adhesive and observe it under a microscope.

[0097] Note: For multiple sequential staining steps, the staining time and actual amount used need to be adjusted and optimized depending on the specific sample being tested and the fully automated immunohistochemical staining machine, in order to achieve the best staining effect.

[0098] The results are as follows Figure 1 As shown, the red staining of Ki67 in lymphoma tissue did not show any color bleeding; the red was bright and contrasted sharply with the brown of CD3.

[0099] Example 5

[0100] In this embodiment, the cross-staining agent used for multiple sequential immunohistochemical staining is applied using a fully automated immunohistochemical staining machine and the cross-staining agent described in Example 2 of this invention. In lymphoma tissue, Ki67 is first stained red, followed by CD3 stained brown, for multiple sequential staining. The specific implementation method is as described in Example 4.

[0101] The results are as follows Figure 2As shown, the red staining of Ki67 in lymphoma tissue did not show any color bleeding; the red was bright and contrasted sharply with the brown of CD3.

[0102] Example 6

[0103] In this embodiment, the cross-staining agent used for multiple sequential immunohistochemical staining is applied using a fully automated immunohistochemical staining machine and the cross-staining agent described in Example 3 of this invention. In lymphoma tissue, Ki67 is first stained red, followed by CD3 stained brown, for multiple sequential staining. The specific implementation method is as described in Example 4.

[0104] The results are as follows Figure 3 As shown, the red staining of Ki67 in lymphoma tissue did not show any color bleeding; the red was bright and contrasted sharply with the brown of CD3.

[0105] Comparative Example 2

[0106] Comparative Example 2 used a fully automated immunohistochemical staining machine and the color-blocking agent of Comparative Example 1. In lymphoma tissue, Ki67 was first stained red, and then CD3 was stained brown, for multiple sequential staining. The specific implementation method is as described in Example 4.

[0107] The results are as follows Figure 4 As shown, the red staining of Ki67 in lymphoma tissue exhibits some color bleeding, resulting in a red-brown mixture to some extent, which contrasts relatively poorly with the brown staining of CD3.

[0108] Comparative Example 3

[0109] Comparative Example 3 used a fully automated immunohistochemical staining machine. In the lymphoma tissue, Ki67 was first stained red, followed by CD3 staining brown. The cross-color blocking agent of this invention was not used; the process was performed normally. The specific implementation method is as follows:

[0110] 1. Dewaxing and hydration of paraffin tissue sections;

[0111] 2. Antigen retrieval: Antigen retrieval buffer II was used to perform high-pressure retrieval on the slides at 100℃ for 20 minutes, with a sample volume of 150ul.

[0112] 3. Add 3% to 3.5% peroxidase blocking agent to block endogenous peroxidase, incubate at room temperature for 5 minutes, and add 150 μL of sample.

[0113] 4. Incubation of the primary antibody in the first procedure: Incubate at 37°C for 30 minutes, with a sample volume of 150 μL.

[0114] 5. Incubation after primary antibody administration in the first procedure: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0115] 6. Incubation of the second antibody in the first procedure: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0116] 7. Rapid red color development: Incubate at room temperature for 8 minutes, and add 150ul of sample.

[0117] 8. Second procedure for primary antibody incubation: Incubate at 37℃ for 30 minutes, with a sample volume of 150ul.

[0118] 9. Incubation after primary antibody administration in the second procedure: Incubate at 37°C for 10 minutes, with a sample volume of 150 μL.

[0119] 10. Second procedure for secondary antibody incubation: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0120] 11. DAB color development: Incubate at room temperature for 5 minutes, and add 150ul of sample.

[0121] 12. Staining: Incubate with hematoxylin at room temperature for 1 minute, and add 150ul of sample.

[0122] 13. Seal the slide with water-based mounting adhesive and observe it under a microscope.

[0123] The results are as follows Figure 5 As shown, the red staining of Ki67 in lymphoma tissue exhibits color bleeding, resulting in a mixed red and brown color that does not contrast sharply with the brown of CD3.

[0124] 2.2 Staining experiments in lung squamous cell carcinoma tissue (see Examples 7-9 and Comparative Examples 4-5)

[0125] In the staining experiments on squamous cell carcinoma tissue of lung, Examples 7-9 and Comparative Examples 4-5 used tissue sections from the same tissue block. As the tissue sections were prepared, the morphology of the tissue in the sections would change due to the irregularity of the tissue morphology, the large experimental volume, and the use of too many sections. This is a normal phenomenon.

[0126] Example 7

[0127] The application of the color-blocking agent in the immunohistochemical multiple sequential staining of this embodiment uses a fully automated immunohistochemical staining machine and the color-blocking agent of Embodiment 1 of the present invention. In lung squamous cell carcinoma tissue, CK5 & 6 are first stained brown, then p40 is stained red, for multiple sequential staining. The specific implementation method is as follows:

[0128] 1. Dewaxing and hydration of paraffin tissue sections;

[0129] 2. Antigen retrieval: Antigen retrieval buffer II was used to perform high-pressure retrieval on the slides at 100℃ for 20 minutes, with a sample volume of 150ul.

[0130] 3. Add 3% to 3.5% peroxidase blocking agent to block endogenous peroxidase, incubate at room temperature for 5 minutes, and add 150 μL of sample.

[0131] 4. Incubation of the primary antibody in the first procedure: Incubate at 37°C for 30 minutes, with a sample volume of 150 μL.

[0132] 5. Incubation after primary antibody administration in the first procedure: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0133] 6. Incubation of the second antibody in the first procedure: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0134] 7. DAB color development: Incubate at room temperature for 5 minutes, and add 150ul of sample.

[0135] 8. Color-blocking agent: Block at 40-60℃ for 10 minutes, with a sample volume of 150ul.

[0136] 9. Incubation of the primary antibody in the second procedure: Incubate at 37°C for 30 minutes, with a sample volume of 150 μL.

[0137] 10. Incubation after primary antibody administration in the second procedure: Incubate at 37°C for 10 minutes, with a sample volume of 150 μL.

[0138] 11. Second procedure for secondary antibody incubation: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0139] 12. Rapid red color development: Incubate at room temperature for 8 minutes, and add 150ul of sample.

[0140] 13. Staining: Incubate with hematoxylin at room temperature for 1 minute, and add 150ul of sample.

[0141] 14. Seal the slide with water-based mounting adhesive and observe it under a microscope.

[0142] The results are as follows Figure 6 As shown, the brown staining of CK5 & 6 in lung squamous cell carcinoma tissue did not show color bleeding, and p40 red was bright, accurately located, surrounded by CK5 & 6, and showed a sharp contrast with the brown staining of CK5 & 6.

[0143] Example 8

[0144] In this embodiment, the cross-staining agent used for multiple sequential immunohistochemical staining was applied using a fully automated immunohistochemical staining machine and the cross-staining agent described in Example 2 of this invention. Multiple sequential staining was performed on lung squamous cell carcinoma tissue, first brown staining of CK5 & 6, then red staining of p40. The specific implementation method is as described in Example 7.

[0145] The results are as follows Figure 7As shown, the brown staining of CK5 & 6 in lung squamous cell carcinoma tissue did not show color bleeding, and p40 red was bright, accurately located, surrounded by CK5 & 6, and showed a sharp contrast with the brown staining of CK5 & 6.

[0146] Example 9

[0147] In this embodiment, the cross-staining agent used for multiple sequential immunohistochemical staining was applied using a fully automated immunohistochemical staining machine and the cross-staining agent described in Example 3 of this invention. Multiple sequential staining was performed on lung squamous cell carcinoma tissue, first brown staining of CK5 & 6, then red staining of p40. The specific implementation method is as described in Example 7.

[0148] The results are as follows Figure 8 As shown, the brown staining of CK5 & 6 in lung squamous cell carcinoma tissue did not show color bleeding, and p40 red was bright, accurately located, surrounded by CK5 & 6, and showed a sharp contrast with the brown staining of CK5 & 6.

[0149] Comparative Example 4

[0150] Comparative Example 4 used a fully automated immunohistochemical staining machine and the color-blocking agent of Comparative Example 1. In lung squamous cell carcinoma tissue, CK5 & 6 were first stained brown, and then p40 was stained red, for multiple sequential staining. The specific implementation method is as described in Example 5.

[0151] The results are as follows Figure 9 As shown, the brown staining of CK5 & 6 in lung squamous cell carcinoma tissue exhibits some color bleeding, with a slight reddish tinge. Furthermore, the p40 red staining is weak, indicating incomplete penetration into the cells and resulting in weaker staining and relatively poor contrast with CK5 & 6.

[0152] Comparative Example 5

[0153] Comparative Example 5 used a fully automated immunohistochemical staining machine. In the squamous cell carcinoma tissue of the lung, CK5 & 6 were first stained brown, followed by p40 stained red. The cross-color blocking agent of this invention was not used; the process was performed normally in sequence. The specific implementation method is as follows:

[0154] 1. Dewaxing and hydration of paraffin tissue sections;

[0155] 2. Antigen retrieval: Antigen retrieval buffer II was used to perform high-pressure retrieval on the slides at 100℃ for 20 minutes, with a sample volume of 150ul.

[0156] 3. Add 3% to 3.5% peroxidase blocking agent to block endogenous peroxidase, incubate at room temperature for 5 minutes, and add 150 μL of sample.

[0157] 4. Incubation of the primary antibody in the first procedure: Incubate at 37°C for 30 minutes, with a sample volume of 150 μL.

[0158] 5. Incubation after primary antibody administration in the first procedure: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0159] 6. Incubation of the second antibody in the first procedure: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0160] 7. DAB color development: Incubate at room temperature for 5 minutes, and add 150ul of sample.

[0161] 8. Second procedure for primary antibody incubation: Incubate at 37℃ for 30 minutes, with a sample volume of 150ul.

[0162] 9. Incubation after primary antibody administration in the second procedure: Incubate at 37°C for 10 minutes, with a sample volume of 150 μL.

[0163] 10. Second procedure for secondary antibody incubation: Incubate at 37℃ for 10 minutes, with a sample volume of 150ul.

[0164] 11. Rapid red color development: Incubate at room temperature for 8 minutes, and add 150ul of sample.

[0165] 12. Staining: Incubate with hematoxylin at room temperature for 1 minute, and add 150ul of sample.

[0166] 13. Seal the slide with water-based mounting adhesive and observe it under a microscope.

[0167] The results are as follows Figure 10 As shown, the brown staining from CK5 & CK6 in lung squamous cell carcinoma tissue exhibited color bleeding, resulting in a mixed reddish-brown color with an overall reddish tint. Furthermore, p40 red staining was very weak, failing to penetrate and showing poor contrast with CK5 & CK6.

[0168] In summary, the use of the cross-color blocking agent of this invention in immunohistochemical multiple sequential staining can effectively solve the cross-color problem, significantly improve the staining effect, prevent mutual interference between the localization staining of several antibodies, and make the results more accurate.

Claims

1. An immunohistochemical multiplex sequential staining with cross-talk blocking agent, characterized by: It mainly includes an oxidant, an eluent, and a buffer; the oxidant is one or more of hydrogen peroxide, sodium percarbonate, and nickel peroxide; the eluent is one or more of sodium dodecyl sulfate, ammonium deoxycholate, sodium cholate, sarcosine, Triton-X100, CHAPS, urea, dodecyl maltodextrin, Tween-20, Tween-80, and digitalis saponins; the mass ratio of the oxidant to the eluent is 0.3–4:0.5–3; the mass content of the oxidant in the color-crossing inhibitor is 0.3–4%.

2. The color-crossing blocking agent according to claim 1, characterized in that: The buffer has a pH of 6.5 to 10.

0.

3. The color-crossing blocking agent according to claim 2, characterized in that: The buffer has a pH of 7.0 to 8.

5.

4. The color-crossing blocking agent according to claim 1, characterized in that: It also includes one, two, or three of the following: stabilizer, defoamer, and preservative; the mass ratio of the oxidant to the stabilizer is 0.1–5:0.1–20; the mass ratio of the oxidant to the defoamer is 0.1–5:0.1–5; and the mass ratio of the oxidant to the preservative is 0.1–5:0.01–1.

5. The color-crossing blocking agent according to claim 4, characterized in that: The stabilizer is one or more of bovine serum albumin, calf serum, glycine, glycerol, and polyethylene glycol 6000-20000; the defoamer is polydimethylsiloxane; and the preservative is any one of Proclin 950, gentamicin sulfate, and Kathon.

6. The color-crossing blocking agent according to claim 1, characterized in that: The amount of buffer used is 5-600 mM.

7. The application of a color-blocking agent as described in any one of claims 1 to 6 in immunohistochemical multiple sequential staining, characterized in that: After the previous staining, incubate with a cross-linking inhibitor to block the cross-linking before proceeding with the next staining.

8. The application of the color-blocking agent according to claim 7 in immunohistochemical multiple serial staining, characterized in that: The incubation blocking temperature is 40-60℃, and the time is 10 minutes.

9. The application of the color-blocking agent according to claim 7 in immunohistochemical multiple serial staining, characterized in that: The immunohistochemical multiple sequential staining is performed manually or fully automated.

Images