Confining liquid and method for reducing background signal in immunohistochemical detection

Abstract

The invention relates to a confining liquid and method for reducing background signals in immunohistochemical detection, and belongs to the technical field of immunodetection, and the confining liquid comprises whey protein and an anionic surfactant. By utilizing the confining liquid provided by the invention, background signals generated on a glass slide can be reduced or even eliminated, and the convenience and accuracy of interpretation of an immunohistochemical detection result are improved.

Description

Technical Field

[0001] This application relates to the field of immunoassay technology, and in particular to a blocking solution and method for reducing background signal in immunohistochemical assays. Background Technology

[0002] Background signal (or nonspecific staining) is a common problem in immunohistochemical testing. It can obscure the true target, leading to difficulties in interpreting results or even errors. The generation of background signal is mainly due to the following reasons: (1) Antibody-related factors, such as non-specific binding of antibodies, cross-reactivity of antibodies, or non-specific binding of secondary antibodies to endogenous immunoglobulins in tissues (such as antibodies in plasma cells) or cells with Fc receptors (such as macrophages and lymphocytes).

[0003] (2) Factors inherent to the tissue sample itself, such as endogenous peroxidase activity, endogenous biotin, tissue autofluorescence, tissue necrosis or edge effect and charge interaction.

[0004] Background signals caused by the above-mentioned factors are all in the tissue and can generally be resolved by selecting antibodies and using blocking agents for blocking.

[0005] However, in practice, besides the background signal appearing in the tissue, some yellow DAB deposits also appear on the slides with the tissue attached. This non-specific staining on the slides causes the background signal to appear, such as... Figure 1 As shown, this will also affect the interpretation of the staining results. Summary of the Invention

[0006] To solve at least one of the above-mentioned technical problems, the technical solution adopted in this application is as follows.

[0007] The first aspect of this application provides the use of whey protein and anionic surfactant in the preparation of blocking solutions for reducing background signals in immunohistochemical detection.

[0008] Immunohistochemistry (IHC) is a research technique that utilizes the principle of specific binding between antigens and antibodies. Through a chemical reaction, a chromogenic agent labeled with antibodies is made colored, thereby allowing for the localization, qualitative analysis, and relative quantification of specific antigens (target proteins) within tissues or cells.

[0009] Those skilled in the art will understand that IHC detection is primarily performed on glass slides: paraffin-embedded tissue sections are attached to glass slides through processes such as mounting and copying. As described in the background section, background signals (i.e., non-specific staining) can be generated during IHC detection for various reasons, one important type of which is the background signal generated on the glass slide.

[0010] In this application, whey protein is used as a protein blocking agent. Compared with the common protein blocking agents used in the prior art, such as serum (including but not limited to normal goat serum, normal donkey serum and fetal bovine serum) and inert proteins (including but not limited to bovine serum albumin and skim milk), this application uses whey protein as a protein blocking agent. Because its isoelectric point is pH 4.0-5.0, it is negatively charged in a weakly alkaline environment and can bind to the positive charge on the surface of the slide, thereby inhibiting the non-specific binding of the positive charge on the slide to other reagents such as primary antibodies. Moreover, throughout the entire immunohistochemical staining process, its reaction environment is always weakly alkaline or neutral, thus better blocking non-specific binding sites on the slide that may generate background signals.

[0011] Surfactants (detergents) are not blocking agents themselves; they are added to the blocking solution to help reduce background. In this application, anionic surfactants such as sodium dodecylbenzenesulfonate are used, which can make the substances in the solution bind more uniformly, and at the same time bind with the positive charge on the surface of the glass slide, neutralizing its polarity and significantly reducing the background signal.

[0012] This application utilizes whey protein and anionic surfactants to seal the glass slide, which has a significant synergistic effect and can eliminate background signals generated on the glass slide.

[0013] A second aspect of this application provides a blocking solution for reducing background signals in immunohistochemical detection, the blocking solution comprising whey protein and anionic surfactant.

[0014] In some embodiments of this application, the anionic surfactant includes, but is not limited to, sodium dodecyl sulfate, sodium dodecyl polyoxyethylene ether sulfate, sodium dodecylbenzene sulfonate, and sodium stearate.

[0015] In some specific embodiments of this application, the anionic surfactant is sodium dodecylbenzenesulfonate.

[0016] In this application, the protein protectant is used to protect the whey protein from damage. In some embodiments of this application, the protein protectant is selected from one or more of the group consisting of sucrose, trehalose, sorbitol, and mannitol. In some specific embodiments of this application, the protein protectant is sucrose. In some specific embodiments of this application, the mass percentage of sucrose in the blocking solution is 2% to 5%. In this application, mass percentage is also called mass-volume fraction, or expressed as "w / v". If the mass percentage of sucrose is 3%, it means that 100 mL of the blocking solution contains 3 g of sucrose.

[0017] In some embodiments of this application, the blocking solution is prepared using one selected from PBS buffer, Tris-HCl buffer, HEPES buffer, and acetate buffer.

[0018] In some specific embodiments of this application, the blocking solution is prepared using PBS buffer, comprising 0.8% sodium chloride, 0.02% potassium chloride, 0.287% disodium hydrogen phosphate, and 0.02% potassium dihydrogen phosphate by mass. In some embodiments of this application, the pH of the PBS buffer is 7.2-7.6.

[0019] In some embodiments of this application, the whey protein in the blocking solution has a mass percentage of 2.5% to 20%, and the anionic surfactant has a mass percentage of 0.125% to 2%.

[0020] A third aspect of this application provides a method for reducing background signal in immunohistochemical detection, the method comprising the following steps during the blocking process after antigen retrieval and before primary antibody incubation: A glass slide containing tissue is sealed using the sealing liquid described in any of the second aspects of this application.

[0021] Immunohistochemical detection methods begin with paraffin-embedded sections and include processes such as dewaxing and hydration, antigen retrieval, blocking, primary antibody incubation, secondary antibody incubation, DAB staining, hematoxylin counterstaining, dehydration and clearing, and mounting.

[0022] The blocking process, also known as the blocking process, is to reduce background signals. As those skilled in the art know, background signals are generated not only on the slide but also primarily in the tissue.

[0023] In some embodiments of this application, the closure process further includes a step of blocking background signals generated in the tissue.

[0024] In some embodiments of this application, the closing process includes at least one of the following steps: (1) Block endogenous peroxidase; (2) Block endogenous biotin; (3) Block components in tissues that can bind nonspecifically to antibodies.

[0025] In this field, 3% hydrogen peroxide is typically used to block endogenous peroxidase, commercially available endogenous biotin inhibitors are used to block endogenous biotin, and normal serum homologous to the secondary antibody (such as 10% normal goat serum) or BSA is used to block the non-specific binding between the antibody and tissue.

[0026] In some specific embodiments of this application, during the sealing process, endogenous peroxidase is first blocked using 3% hydrogen peroxide, and then the glass slide is sealed using the sealing solution.

[0027] In some specific embodiments of this application, the sealing time of the glass slide using the sealing liquid is 5 to 15 minutes, for example, 10 minutes.

[0028] Compared with the prior art, this application has the following advantages: In immunohistochemical testing, those skilled in the art typically focus only on background signals generated within the tissue, such as nonspecific staining caused by endogenous peroxidase. Background signals generated on the slide are often overlooked. However, the slide is a crucial substrate for supporting the tissue, and nonspecific staining on it can significantly affect the interpretation of results. During actual operation, the inventors of this application unexpectedly discovered that using whey protein and anionic surfactant to seal the slide effectively eliminates background signals on the slide.

[0029] The blocking solution of this application can reduce or even eliminate background signals generated on the slide, thereby improving the convenience and accuracy of interpreting immunohistochemical test results.

[0030] It should be understood that the description in this section is not intended to identify key or essential features of the embodiments of this application, nor is it intended to limit the scope of this application. Other features of this application will become readily apparent from the following description. Attached Figure Description

[0031] The above and other objects, features, and advantages of exemplary embodiments of this application will become readily apparent from the following detailed description taken in conjunction with the accompanying drawings. Several embodiments of this application are illustrated in the drawings by way of example and not limitation, in which: Figure 1 The background signal generated on the slide when the slide is not sealed is shown; Figure 2 This illustrates the background signal generated on the glass slide after sealing it with sealing liquids #1 to #5 in Embodiment 1 of this application. Figure 3 This illustrates the background signal generated on the glass slide after sealing it with sealing liquid #6~#10 in Embodiment 2 of this application. Figure 4 This illustrates the background signal generated on the glass slide after sealing it with sealing liquid #11~#16 in Embodiment 3 of this application. Figure 5The illustration shows the background signal generated on the glass slide after sealing it with sealing liquid #17~#19 in Embodiment 4 of this application.

[0032] Figure 6 The illustration shows the background signal generated on the slide after sealing it with sealing liquid #20~#22 in Embodiment 4 of this application. Detailed Implementation

[0033] Unless otherwise stated, implied from the context, or as is customary in the art, all parts and percentages in this application are based on weight, and all testing and characterization methods used are concurrent with the filing date of this application. Where applicable, any patent, patent application, or disclosure relating to this application is incorporated herein by reference in its entirety, and its equivalent patent families are also incorporated herein by reference, particularly the definitions of relevant terms in the art disclosed in such documents. If any definition of a specific term disclosed in the prior art is inconsistent with any definition provided in this application, the definition provided in this application shall prevail.

[0034] The numerical ranges used in this application are approximate values ​​and therefore may include values ​​outside the range unless otherwise stated. The numerical range includes all values ​​from the lower limit to the upper limit, increasing by one unit, provided that there is an interval of at least two units between any lower and any higher value. For ranges containing values ​​less than 1 or fractions greater than 1 (e.g., 1.1, 1.5, etc.), one unit is appropriately considered as 0.0001, 0.001, 0.01, or 0.1. For ranges containing single digits less than 10 (e.g., 1 to 5), one unit is generally considered as 0.1. These are merely specific examples of what is intended to be expressed, and all possible combinations of values ​​between the listed minimum and maximum values ​​are considered to be clearly described in this application.

[0035] The terms “comprising,” “including,” “having,” and their derivatives do not exclude the presence of any other components, steps, or processes, regardless of whether such other components, steps, or processes are disclosed in this application. To eliminate any doubt, unless expressly stated otherwise, all compositions using the terms “comprising,” “including,” or “having” in this application may contain any additional additives, excipients, or compounds. Conversely, except for those necessary for operational performance, the term “substantially constitutes…” excludes any other components, steps, or processes described below with respect to that term. The term “consisting of…” does not include any components, steps, or processes not specifically described or listed. Unless expressly stated otherwise, the term “or” refers to the individual members listed or any combination thereof.

[0036] To make the technical problems, technical solutions and beneficial effects solved by this application clearer, the following detailed description is provided in conjunction with embodiments.

[0037] The following examples are used to illustrate preferred embodiments of this application. Those skilled in the art will understand that the techniques disclosed in the examples represent technologies discovered by the inventors that can be used to implement this application, and therefore can be considered preferred embodiments of this application. However, those skilled in the art should understand from this specification that many modifications can be made to the specific embodiments disclosed herein, still yielding the same or similar results, without departing from the spirit or scope of this application.

[0038] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application pertains, and all materials cited herein and referenced by them are incorporated herein by reference.

[0039] Those skilled in the art will recognize, or can learn through routine experimentation, many equivalents of the specific embodiments of the invention described herein. These equivalents will be included in the claims.

[0040] Unless otherwise specified, the experimental methods used in the following examples are conventional methods. Unless otherwise specified, the instruments and equipment used in the following examples are all conventional laboratory instruments and equipment; unless otherwise specified, the experimental materials used in the following examples were all purchased from conventional biochemical reagent stores.

[0041] Example 1: Preparation and application of sealing liquid The inventors conducted an in-depth analysis of the causes of background staining during immunohistochemical testing and found that it was due to the following reasons: To improve tissue adhesion, the slide surface is typically treated with poly-L-lysine or by applying a charge, making it less prone to detachment during staining. However, this treatment inevitably causes the slide to absorb the primary antibody, post-primary antibody reagent, or secondary antibody reagent used in the staining process, resulting in a yellow background on the slide during development.

[0042] When using fully automated immunohistochemical staining equipment, the cleaning process is relatively gentle and cannot completely wash away the components of the primary antibody reagent, post-primary antibody reagent, or secondary antibody reagent adhering to the slide, resulting in a more severe yellowing of the slide after staining.

[0043] Based on the above analysis results, the inventors attempted to use a blocking solution to block the residual reagents on the glass slides, thereby reducing non-specific staining.

[0044] 1. Preparation of sealing solution This embodiment provides five blocking solutions #1 to #5, prepared using PBS buffer (which also serves as a control blocking solution, #0). The PBS buffer, per 10000 mL, comprises 80 g NaCl, 2 g KCl, 28.7 g Na₂HPO₄, and 2 g KH₂PO₄. The preparation method is as follows: accurately weigh NaCl, KCl, Na₂HPO₄, and KH₂PO₄ into a clean glass container, add 8000 mL of purified water and mix thoroughly. Adjust the pH to 7.2-7.6 with dilute hydrochloric acid, and then bring the volume to 10000 mL using deionized water.

[0045] Blocking solutions #1 through #5 are all based on PBS buffer with the same mass of protein protectant (sucrose in this example) and whey protein containing different mass fractions (1%, 2.5%, 5%, 10%, 20%). The specific formulations are shown in Table 1. Table 1: Formulations containing whey protein #1~#5 blocking solution

[0046] The specific preparation method for blocking solutions #1 to #5 is as follows: accurately weigh sucrose and whey protein into a clean container, add 80 mL of prepared PBS buffer, and after the solid substances are fully dissolved, continue to add PBS buffer and adjust the volume to 100 mL.

[0047] 2. Application of blocking solutions #1~#5 in immunohistochemical detection Blocking solutions #1 to #5 were applied to PMS2 immunohistochemical detection (tissue was colon tissue, including cancerous and normal tissue), with PBS buffer (blocking solution #0) as a control.

[0048] The reagents used for PMS2 immunohistochemical detection are shown in Table 2.

[0049] Table 2: Reagent information for PMS2 immunohistochemical assay

[0050] The immunohistochemical detection steps are as follows: ① Baking: Bake the slices in a 62℃ oven for 30 minutes; ② Dewaxing and hydration: Dewaxing and hydration with anhydrous ethanol, 95% and 75% graded alcohols; ③ Antigen retrieval: Retrieve antigens in EDTA antigen retrieval solution under high temperature and high pressure (120℃, 103kPa) for 2 minutes, then allow to cool naturally; ④ Endogenous peroxidase blocking: Block with 3% hydrogen peroxide for 10 min; ⑤ Slide sealing: Immerse the slides directly in a staining box containing #0-#5 sealing solution for 10 minutes to seal the slide background; ⑥ Primary antibody incubation: Add PMS2 primary antibody reagent and incubate for 30 min; ⑦ Secondary antibody incubation: Add polymer secondary antibody reagent and incubate for 30 min; ⑧ DAB color development: Add DAB color developer and incubate for 3 minutes; ⑨ Hematoxylin counterstaining: Counterstain for 7 minutes, then rinse with washing solution for 10 seconds to regain blue color; ⑩ Dehydration and Transparency: Gradient alcohol dehydration results in a transparent dewaxing solution; ⑪ Mounting: Add neutral resin to mount the slide, then inspect it.

[0051] Staining results as follows Figure 2 As shown, according to Figure 2 The results were described using a scale of 0 to 3 to indicate the degree of staining on the slide background: "0" indicates a clean slide with no yellow background; "1" indicates a weak, light yellow background; "2" indicates a relatively obvious yellow background; and "3" indicates a very dark yellow background on the slide. The results are shown in Table 3.

[0052] Table 3: Background staining of glass slides after sealing with sealing solutions #1~#5

[0053] Depend on Figure 2 As shown in Table 3, in PMS2 immunohistochemical assays, blocking the slides with PBS buffer failed to eliminate the yellow background, resulting in a staining degree of "3". Blocking with #1 blocking solution lightened the yellow background color, achieving a staining degree of "2", but the effect was not ideal; the blocking effect did not improve with increasing whey protein content.

[0054] Example 2: In the blocking solution, whey protein was replaced with sodium dodecyl sulfonate. Given that the sealing effect of blocking solutions #1 to #5 on the glass slides in Example 1 was not ideal, this example provides five additional blocking solutions, numbered #6 to #10. Blocking solutions #6 to #10 are all based on the PBS buffer (#0 blocking solution) prepared in Example 1, with the same mass of sucrose added, and sodium dodecyl sulfate containing different mass fractions (0.125%, 0.25%, 0.5%, 1%, and 2%), as shown in Table 4. Table 4: Formulations of sealing solutions containing sodium dodecyl sulfonate #6~#10

[0055] The specific preparation method for blocking solutions #6~#10 is as follows: accurately weigh sucrose and sodium dodecyl sulfonate into a clean container, add 80 mL of prepared PBS buffer, and after the solid substances are fully dissolved, continue to add PBS buffer and adjust the volume to 100 mL.

[0056] Similarly, blocking solutions #6 to #10 were applied to PMS2 immunohistochemical assays, with PBS buffer (#0 blocking solution) as a control.

[0057] The reagents used for PMS2 immunohistochemical detection are the same as in Example 1; the immunohistochemical detection method is the same as in Example 1, except that the sealing agent used on the slides is different.

[0058] Staining results as follows Figure 3 As shown, according to Figure 3 The results show that the degree of staining of the slide background was described using a scale of 0 to 3, as shown in Table 5.

[0059] Table 5: Background staining of glass slides after sealing with sealing solutions #6~#10

[0060] Depend on Figure 3 As shown in Table 5, replacing whey protein with sodium dodecyl sulfate in Example 1 resulted in a better blocking effect than PBS buffer, but no improvement compared to whey protein.

[0061] Example 3: The blocking solution composition includes both whey protein and sodium dodecyl sulfonate. Given that the blocking solutions #1 to #5 in Example 1 and #6 to #10 in Example 2 did not provide ideal blocking effects on the glass slides, this example provides six additional blocking solutions, numbered #11 to #16. Blocking solutions #11 to #16 are all based on the PBS buffer (#0 blocking solution) prepared in Example 1, with the same mass of sucrose added, along with different mass fractions (0.125%, 0.25%, 0.5%, 1%) of sodium dodecyl sulfate and different mass fractions (2.5%, 5%) of whey protein. The specific formulations are shown in Table 6. Table 6: Formulations of Blocking Fluids Containing Whey Protein and Sodium Dodecyl Sulfate #11~#16

[0062] The specific preparation method for blocking solutions #11~#16 is as follows: accurately weigh sucrose, whey protein and sodium dodecyl sulfate into a clean container, add 80 mL of prepared PBS buffer, and after the solid substances are fully dissolved, continue to add PBS buffer and adjust the volume to 100 mL.

[0063] Similarly, blocking solutions #11 to #16 were used in PMS2 immunohistochemical assays, with PBS buffer (#0 blocking solution) as a control.

[0064] The reagents used for PMS2 immunohistochemical detection are the same as in Example 1; the immunohistochemical detection method is the same as in Example 1, except that the sealing agent used on the slides is different.

[0065] Staining results as follows Figure 4 As shown, according to Figure 4 The results show that the degree of staining of the slide background was described using a scale of 0 to 3, as shown in Table 7.

[0066] Table 7: Background staining of glass slides after sealing with sealing solutions #11~#16

[0067] Depend on Figure 4 As shown in Table 7, the sealing effect on the slides was significantly improved when whey protein and sodium dodecyl sulfate were included in the sealing solution. Furthermore, when the sodium dodecyl sulfate was increased to more than 0.5% w / v, no yellow color appeared on the slides, and the background staining degree was "0", indicating that the background signal had been completely eliminated and the sealing effect on the slides was very good.

[0068] Furthermore, in conjunction with Examples 1, 2, and 3, it was found that: When the blocking solution contains 5% w / v whey protein (#3 blocking solution), the background staining level is "2"; when the blocking solution contains 1% w / v sodium dodecyl sulfate (#9 blocking solution), the background staining level is also "2"; and when the blocking solution contains 2.5% w / v whey protein and 0.5% w / v sodium dodecyl sulfate (#13 blocking solution), the background staining level is "0".

[0069] When the blocking solution contains 10% w / v whey protein (#4 blocking solution), the background staining level is "2"; when the blocking solution contains 1% w / v sodium dodecyl sulfate (#9 blocking solution), the background staining level is also "2"; and when the blocking solution contains 5% w / v whey protein and 0.5% w / v sodium dodecyl sulfate (#14 blocking solution), the background staining level is "0".

[0070] When the blocking solution contains 5% w / v whey protein (#3 blocking solution), the background staining level is "2"; when the blocking solution contains 2% w / v sodium dodecyl sulfate (#10 blocking solution), the background staining level is also "2"; and when the blocking solution contains 2.5% w / v whey protein and 1% w / v sodium dodecyl sulfate (#15 blocking solution), the background staining level is "0".

[0071] When the blocking solution contains 10% w / v whey protein (#4 blocking solution), the background staining level is "2"; when the blocking solution contains 2% w / v sodium dodecyl sulfate (#10 blocking solution), the background staining level is also "2"; and when the blocking solution contains 5% w / v whey protein and 1% w / v sodium dodecyl sulfate (#16 blocking solution), the background staining level is "0".

[0072] All of the above results indicate that whey protein and sodium dodecyl sulfate have a synergistic effect on the sealing of glass slides.

[0073] Example 4: Replacing whey protein with BSA in the blocking solution component To further verify the synergistic effect of whey protein and sodium dodecyl sulfate, this embodiment provides three additional blocking solutions, numbered #17 to #19. Blocking solutions #17 to #19 are all based on the PBS buffer (#0 blocking solution) prepared in Example 1, with the same mass of sucrose and the same mass of sodium dodecyl sulfate, and different mass fractions (2.5%, 5%, and 10%) of BSA. The specific formulations are shown in Table 8. Table 8: Formulations of sealing solutions containing BSA and sodium dodecyl sulfonate #17~#19

[0074] The specific preparation method for blocking solutions #17~#19 is as follows: accurately weigh sucrose, BSA and sodium dodecyl sulfonate into a clean container, add 80 mL of prepared PBS buffer, and after the solid substances are fully dissolved, continue to add PBS buffer and adjust the volume to 100 mL.

[0075] Similarly, blocking solutions #17 to #19 were applied to PMS2 immunohistochemical assays. The reagents used for PMS2 immunohistochemical assays were the same as in Example 1; the immunohistochemical assay method was the same as in Example 1, except that the blocking agent used on the slides was different.

[0076] Staining results as follows Figure 5 As shown, according to Figure 5 The results show that the degree of staining of the slide background was described using a scale of 0 to 3, as shown in Table 9.

[0077] Table 9: Background staining of glass slides after sealing with sealing solutions #17~#19

[0078] Depend on Figure 5 As shown in Table 9, if the combination of whey protein and sodium dodecyl sulfonate is replaced with the combination of BSA and sodium dodecyl sulfonate, the blocking effect disappears, and the background staining degree of the slides is "3", which further proves that whey protein and sodium dodecyl sulfonate have a synergistic effect.

[0079] Example 5: Replacing sodium dodecyl sulfonate with Tween-20 in the sealing liquid component. To further verify the synergistic effect of whey protein and sodium dodecyl sulfate, this embodiment provides three additional blocking solutions, numbered #20 to #22. Blocking solutions #20 to #22 are all based on the PBS buffer (#0 blocking solution) prepared in Example 1, with the same mass of sucrose and whey protein, and different volume fractions (0.5%, 1%, and 2%) of Tween-20. The specific formulations are shown in Table 10. Table 10: Formulations of #20~#22 blocking solutions containing whey protein and Tween-20

[0080] The specific preparation method for blocking solutions #20~#22 is as follows: accurately weigh / measure sucrose, whey protein and Tween-20 into a clean container, add 80mL of prepared PBS buffer, and after the solid substances are fully dissolved, continue to add PBS buffer and adjust the volume to 100mL.

[0081] Similarly, blocking solutions #20 to #22 were applied to PMS2 immunohistochemical assays. The reagents used for PMS2 immunohistochemical assays were the same as in Example 1; the immunohistochemical assay method was the same as in Example 1, except that the blocking agent used on the slides was different.

[0082] Staining results as follows Figure 6 As shown, according to Figure 6 The results show that the degree of staining of the slide background was described using a scale of 0 to 3, as shown in Table 11.

[0083] Table 11: Background staining of glass slides after sealing with sealing solutions #20~#22

[0084] Depend on Figure 6 As shown in Table 11, if the combination of whey protein and sodium dodecyl sulfate is replaced with the combination of whey protein and Tween-20, the blocking effect disappears, and the background staining degree of the slides is "3", which further proves that whey protein and sodium dodecyl sulfate have a synergistic effect.

[0085] None of the above experiments had a negative impact on the immunohistochemical staining results. The interpretation results of all blocked slides were consistent with those of untreated slides, and the interpretation was easier than that of untreated slides.

[0086] Further analysis by the inventors revealed that a key reason for the effectiveness of sodium dodecyl sulfate is its role as an anionic surfactant, enabling it to bind with the positive charge on the surface of the glass slide and neutralize its polarity. In other words, other anionic surfactants achieve the same technical effect as sodium dodecyl sulfate.

[0087] Furthermore, it should be understood that after reading the foregoing teachings of this application, those skilled in the art can make various alterations or modifications to this application, and these equivalent forms also fall within the scope defined by the appended claims.

Claims

1. Application of whey protein and anionic surfactant in the preparation of blocking solutions for reducing background signal in immunohistochemical detection.

2. A blocking solution for reducing background signal in immunohistochemical detection, characterized in that, The sealing solution includes whey protein and anionic surfactant.

3. The sealing liquid according to claim 2, characterized in that, The anionic surfactant is sodium dodecylbenzenesulfonate.

4. The sealing liquid according to claim 3, characterized in that, The sealing solution also includes a protein protectant.

5. The sealing liquid according to claim 4, characterized in that, The protein protectant is selected from one or more of the group consisting of sucrose, trehalose, sorbitol and mannitol.

6. The sealing liquid according to any one of claims 2 to 5, characterized in that, The blocking solution is prepared using one of the following: PBS buffer, Tris-HCl buffer, HEPES buffer, and acetate buffer.

7. The sealing liquid according to claim 6, characterized in that, In the blocking solution, the whey protein has a mass percentage of 2.5% to 20%; and the surfactant has a mass percentage of 0.125% to 2%.

8. A method for reducing background signal in immunohistochemical detection, characterized in that, The blocking process after antigen retrieval and before primary antibody incubation includes the following steps: A glass slide containing tissue is sealed using the sealing liquid described in any one of claims 2 to 7.

9. The method according to claim 8, characterized in that, The sealing process also includes a step of blocking background signals generated in the tissue.

10. The method according to claim 9, characterized in that, The closure process includes at least one of the following steps: (1) Block endogenous peroxidase; (2) Block endogenous biotin; (3) Block components in tissues that can bind nonspecifically to antibodies.

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