DAB immunohistochemical staining decoloring solution and decoloring method

Abstract

This invention discloses a destaining solution for DAB immunohistochemical staining. The destaining solution comprises a functionalized ionic liquid with a volume concentration of 10%-40% and a co-solvent of 60%-90%. The functionalized ionic liquid is a carboxyl-functionalized imidazole ionic liquid and / or a hydroxypyridine ionic liquid. The co-solvent is a glycerol solution. The destaining solution of this application uses a functionalized ionic liquid as the core active component, which can rapidly and thoroughly remove DAB chromogenic products. The ionic liquid has good biocompatibility, and when combined with a specific concentration of glycerol co-solvent, it can create a moisturizing environment, effectively preventing tissue drying and detachment. The mild pH environment and the selection of a neutral protease ensure that the tissue morphology and antigen activity are not damaged, facilitating subsequent counterstaining operations.

Description

Technical Field

[0001] This invention belongs to the field of immunohistochemistry technology, specifically relating to a DAB immunohistochemistry staining and destaining solution and a destaining method. Background Technology

[0002] DAB, or 3,3-N-Diaminobenzidine Tertrahydrochloride, is the most sensitive and commonly used substrate for horseradish peroxidase. Under the catalysis of horseradish peroxidase, DAB produces a brown precipitate.

[0003] In the fields of pathological diagnosis and scientific research, immunohistochemistry (IHC) staining is one of the most widely used detection methods. It utilizes the specific binding reaction between antigen and antibody, employing DAB (3,3'-diaminobenzidine) chromogenic agent to form a brownish-yellow precipitate on tissue sections to indicate the presence and location of the target antigen. However, in practical applications, it is often necessary to perform multiple staining on the same tissue section or to salvage sections that have failed to stain. This necessitates the removal of the original DAB chromogenic product beforehand.

[0004] Existing DAB destaining methods are mainly divided into two categories: The first category is the strong oxidant method, such as the combination of potassium permanganate and oxalic acid, or the high-concentration hydrogen peroxide system. Although the destaining speed is fast, the strong oxidizing properties can easily damage tissue morphology and antigen sites, leading to weakening or even disappearance of subsequent restaining signals. In addition, the reagents are highly corrosive and have poor safety. The second category is the organic solvent gradient method, such as xylene-ethanol gradient dewaxing and destaining. This method is cumbersome and time-consuming. Xylene is toxic and can easily cause environmental pollution. It also increases the risk of tissue detachment.

[0005] However, existing DAB destaining methods have problems such as difficulty in balancing destaining efficiency, antigen retention capacity, environmental safety, and ease of operation, and cannot meet the needs of pathology laboratories for efficient, green, and low-damage destaining. Summary of the Invention

[0006] To address at least one of the above problems, the present invention provides a DAB immunohistochemical staining destaining solution and a destaining method.

[0007] To achieve the above objectives, the present invention employs the following technical means: The first aspect of the present invention provides a DAB immunohistochemical staining destaining solution, wherein the destaining solution comprises a functionalized ionic liquid with a volume concentration of 10%-40% and a cosolvent of 60%-90%. The functionalized ionic liquid is a carboxyl-functionalized imidazole ionic liquid and / or a hydroxypyridine ionic liquid; The co-solvent is a glycerol solution.

[0008] In some embodiments of the present invention, the functionalized ionic liquid is one or a mixture of two of 1-ethyl-3-methylimidazolium sulfate and N-ethylpyridine tetrafluoroborate.

[0009] In some embodiments of the present invention, the mass concentration of glycerol in the cosolvent is 5%.

[0010] In some embodiments of the present invention, the pH of the decolorizing solution is 6.8-7.5.

[0011] In some embodiments of the present invention, the decolorizing solution further includes an enzymatic hydrolysis component with a volume concentration of 0.05%-0.2%.

[0012] In some embodiments of the present invention, the mild enzymatic hydrolysate is a neutral protease.

[0013] The introduction of enzymatic hydrolysis components in the protocol helps DAB dissociate from the tissue, further improving decolorization efficiency and shortening decolorization time; at the same time, it also avoids damage to antigen sites and tissue morphology, ensuring the signal intensity of subsequent restaining.

[0014] A second aspect of the present invention provides a method for DAB immunohistochemical staining and destaining using the destaining solution described in the first aspect, comprising the following steps: S1. Take the pathological sections after DAB staining and demount them in xylene solution; S2. Completely cover the tissue area with the decolorizing solution described in the first aspect, incubate at room temperature, and observe under a microscope until the DAB brownish-yellow color completely disappears. S3. Rinse the slices with deionized water to completely remove the DAB color.

[0015] In some embodiments of the present invention, in step S1, the slide is desealed in xylene solution for 10-20 minutes.

[0016] In some embodiments of the present invention, in step S2, the greenhouse incubation time is 5-10 minutes. During this incubation time, while ensuring complete decolorization, the risk of tissue damage that may result from prolonged processing can be avoided.

[0017] In some embodiments of the present invention, in step S3, the method of rinsing the slices with deionized water is to rinse 2-3 times, each time for at least 1 minute.

[0018] Beneficial effects of the present invention Compared with the prior art, the present invention has the following beneficial effects: The destaining solution of this application uses a functionalized ionic liquid as the core active component. Through a gentle chemical bond breaking mechanism, and with the assistance of a solubilizer, it protects tissue morphology and aids in penetration. The synergistic effect of these two factors enables rapid and thorough removal of DAB chromogenic products. Furthermore, the ionic liquid exhibits excellent biocompatibility, and when combined with a specific concentration of glycerol as a solubilizer, it creates a moisturizing environment, effectively preventing tissue drying and detachment. The mild pH environment and the selection of a neutral protease, while assisting in the dissociation of DAB, shortening destaining time, and improving destaining efficiency, do not excessively digest tissue structural proteins or damage antigenic epitopes, thus ensuring the feasibility and accuracy of subsequent counterstaining.

[0019] The decolorizing solution does not contain toxic or harmful strong oxidants or organic solvents, making it environmentally friendly and safe, thus reducing the risk of environmental pollution and health hazards to operators.

[0020] The proposed solution is easy to operate. With the help of a specific decolorizing solution, it achieves an efficient and convenient decolorization process. The decolorization process can be carried out at room temperature. The steps are simple and do not require complex gradient treatment, which significantly shortens the operation time and improves the working efficiency of the pathology laboratory. Attached Figure Description

[0021] Figure 1 This is a comparison chart of the decolorization effects of six groups of decolorizing solutions prepared with different concentrations of 1-ethyl-3-methylimidazolium sulfate in Example 1 of the present invention. Figure 2 This is a comparison chart of the decolorization effects of six groups of decolorizing solutions prepared with different concentrations of N-ethylpyridine tetrafluoroborate in Example 2 of the present invention; Figure 3 This is a comparison chart of the decolorization effects of five groups of decolorizing solutions prepared with different concentrations of neutral protease in Example 3 of the present invention; Figure 4 This is a comparison chart of the decolorization effects of three groups of decolorizing solutions with different pH values ​​in Example 4 of the present invention; Figure 5 This is a comparison chart of the decolorization effects of three groups of decolorizing solutions with different concentrations of ionic liquids in Example 5 of the present invention; Figure 6 This is a comparison diagram of the effects of potassium permanganate-oxalic acid decolorization before and after in Comparative Example 1 of the present invention; Figure 7 This is a comparison diagram of the effects of 3% hydrogen peroxide-methanol as a decolorizing solution before and after decolorization in Comparative Example 2 of this invention; Figure 8 This is a comparison diagram of the restaining effects of Scheme 1 and Scheme 2 in Embodiment 6 of the present invention. Detailed Implementation

[0022] The following examples are used to illustrate preferred embodiments of the invention. Those skilled in the art will understand that the techniques disclosed in the examples represent techniques discovered by the inventors that can be used to implement the invention, and therefore can be considered preferred embodiments for implementing the invention. 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 the invention.

[0023] 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 invention pertains, and all materials disclosed herein are incorporated herein by reference. Many equivalent techniques of specific embodiments of the invention described herein will be recognized or can be understood by those skilled in the art through conventional experimentation. These equivalents will be included in the claims.

[0024] The technical solution of this application will be further described in detail below with reference to specific embodiments.

[0025] Example 1 Colorectal cancer tissue stained with CK pan was selected as the destaining solution for functionalized imidazole ionic liquid (1-ethyl-3-methylimidazolium sulfate methyl ester). Six groups of destaining solutions were prepared with different mass concentrations of 1-ethyl-3-methylimidazolium sulfate methyl ester. The destaining effect of different concentrations of 1-ethyl-3-methylimidazolium sulfate methyl ester destaining solutions was compared on the unmounted tissue sections. The incubation temperature of the destaining solutions was room temperature. The 1-ethyl-3-methylimidazolium sulfate methyl ester, product code: Q-0354756, was purchased from Xi'an Qiyue Biotechnology. The specific preparation ratios of the reagents are shown in Table 1 below.

[0026] Table 1. Preparation of decolorizing solutions of 1-ethyl-3-methylimidazolium sulfate at different concentrations (100 mL)

[0027] Note: In the 40% solution, the solute was not completely dissolved, and a small amount precipitated out.

[0028] After the reagents are prepared, proceed with the following steps: 1. Place the DAB-developed paraffin sections of colon cancer into xylene solution for 15 minutes to dissolve the mounting medium, then remove the coverslip; 2. Place the slides with the coverslip removed into the decolorizing solutions of different concentrations mentioned above and let them stand for 10 minutes. 3. Remove the decolorized slices and rinse them three times with deionized water, one minute each time; 4. After drying the slide with a hairdryer, add a mounting medium, cover with a coverslip, and observe the results under a microscope.

[0029] The test results are shown in Table 2 and Figure 1 As shown.

[0030] Table 2 Detection Results

[0031] The results showed that when the concentration of 1-ethyl-3-methylimidazolium sulfate methyl ester salt was between 10% and 40%, incubation at room temperature for 10 minutes could completely decolorize the developed DAB, but 40% 1-ethyl-3-methylimidazolium sulfate methyl ester salt showed slight turbidity.

[0032] Example 2 Colorectal cancer tissue stained with CK pan was selected as the destaining solution for pyridine-based ionic liquid (N-ethylpyridine tetrafluoroborate). Destaining solutions were prepared according to different mass concentrations of N-ethylpyridine tetrafluoroborate. The destaining effect of different concentrations of N-ethylpyridine tetrafluoroborate destaining solutions was compared on the tissue sections after unmounting. The incubation temperature of the destaining solution was room temperature. The N-ethylpyridine tetrafluoroborate product code was Q-0020260, purchased from Xi'an Qiyue Biotechnology. The specific preparation ratios are shown in Table 3 below.

[0033] Table 3. Preparation of decolorizing solutions of N-ethylpyridine tetrafluoroborate at different concentrations (100 mL)

[0034] After the reagents are prepared, proceed with the following steps: 1. Place the DAB-developed paraffin sections of colon cancer into xylene solution for 15 minutes to dissolve the mounting medium, then remove the coverslip; 2. Place the slides with the coverslip removed into the decolorizing solutions of different concentrations mentioned above and let them stand for 10 minutes. 3. Remove the decolorized slices and rinse them three times with deionized water, one minute each time; 4. After drying the slide with a hairdryer, add a mounting medium, cover with a coverslip, and observe the results under a microscope.

[0035] The test results are shown in Table 4 and Figure 2 As shown.

[0036] Table 4 Test Results

[0037] The results showed that when the concentration of N-ethylpyridine tetrafluoroborate was between 10% and 40%, incubation at room temperature for 10 minutes could completely decolorize the developed DAB.

[0038] Example 3 Colon cancer tissue stained with CK pan was selected as the destaining solution for destaining sections. A destaining solution containing 20% ​​1-ethyl-3-methylimidazolium sulfate was used as the base destaining solution. Different mass concentrations of neutral protease (dispersant enzyme) were added. The effect of different concentrations of neutral protease (dispersant enzyme) on the destaining efficiency was compared on the tissue sections after unmounting. The incubation temperature of the destaining solution was room temperature. The neutral protease was A002100, purchased from Shanghai Sangon Biotech.

[0039] The specific mixing ratios are shown in Table 5 below.

[0040] Table 5. Preparation of decolorizing solutions with different concentrations of neutral protease (dispersant enzyme) (100 mL)

[0041] After the reagents are prepared, proceed with the following steps: 1. Place the DAB-developed paraffin sections of colon cancer into xylene solution for 15 minutes to dissolve the mounting medium, then remove the coverslip; 2. Place the slides with the coverslip removed into the decolorizing solutions of different concentrations mentioned above and let them stand for 5 minutes; 3. Remove the decolorized slices and rinse them three times with deionized water, one minute each time; 4. After drying the slide with a hairdryer, add a mounting medium, cover with a coverslip, and observe the results under a microscope.

[0042] The test results are shown in Table 6 and Figure 3 As shown.

[0043] Table 6 Test Results

[0044] The results showed that adding 0.05%-0.2% neutral protease (dispersant) destaining solution to a destaining solution containing 20% ​​1-ethyl-3-methylimidazolium sulfate could completely destain DAB staining within 5 minutes at room temperature without affecting tissue morphology. However, increasing the concentration of neutral protease (dispersant) to 0.4% would affect tissue morphology.

[0045] Example 4 Endometrial cancer tissue stained with CK pan was selected as the destaining solution for decolorization. A destaining solution containing 20% ​​1-ethyl-3-methylimidazolium sulfate was used. The pH of the destaining solution was adjusted to three groups of 6.8, 7.2 and 7.5 respectively. The effect of different pH values ​​of the destaining solution on the destaining effect of DAB stained sections was compared on the tissue sections after unmounting. The incubation temperature of the destaining solution was room temperature. The specific preparation ratios are shown in Table 7 below.

[0046] Table 7. Preparation of decolorizing solutions at different pH values ​​(100 mL)

[0047] After the reagents are prepared, proceed with the following steps: 1. Place the DAB-developed paraffin sections of colon cancer into xylene solution for 15 minutes to dissolve the mounting medium, then remove the coverslip; 2. Place the slides with the coverslip removed into the decolorizing solutions of different concentrations mentioned above and let them stand for 10 minutes. 3. Remove the decolorized slices and rinse them three times with deionized water, one minute each time; 4. After drying the slide with a hairdryer, add a mounting medium, cover with a coverslip, and observe the results under a microscope.

[0048] The test results are shown in Table 8 and Figure 4 As shown.

[0049] Table 8 Test Results

[0050] The results showed that by comparing the effects of pH 6.8-pH 7.5 on the decolorization effect of the decolorizing solution, it was found that within this pH range, it had no effect on the decolorization effect of paraffin tissue sections developed by DAB.

[0051] Example 5 Endometrial cancer tissue stained with CK pan was selected as the destaining solution for destaining sections. 1-Ethyl-3-methylimidazolium sulfate methyl ester salt and N-ethylpyridine tetrafluoroborate were mixed in equal proportions to prepare the destaining solution. The pH of the destaining solution was adjusted to 7.2. The effect of the destaining solution, which was a mixture of two different ionic liquids, on the destaining effect of DAB-stained sections was observed on the tissue sections after unmounting. The incubation temperature of the destaining solution was room temperature. The specific preparation ratios are shown in Table 9 below.

[0052] Table 9. Preparation of decolorizing solutions with different concentrations of ionic liquids (100 mL)

[0053] After the reagents are prepared, proceed with the following steps: 1. Place the DAB-developed paraffin sections of colon cancer into xylene solution for 15 minutes to dissolve the mounting medium, then remove the coverslip; 2. Place the slides with the coverslip removed into the decolorizing solutions of different concentrations mentioned above and let them stand for 10 minutes. 3. Remove the decolorized slices and rinse them three times with deionized water, one minute each time; 4. After drying the slide with a hairdryer, add a mounting medium, cover with a coverslip, and observe the results under a microscope.

[0054] The test results are shown in Table 10 and Figure 5 As shown.

[0055] Table 10 Test Results

[0056] The results showed that the decolorizing solution prepared by mixing two different ionic liquids, 1-ethyl-3-methylimidazolium sulfate and N-ethylpyridine tetrafluoroborate, could completely remove the DAB staining from paraffin tissue sections.

[0057] Comparative Example 1 Colorectal cancer tissue stained with CK pan was selected as the destained sections using the standard potassium permanganate-oxalic acid method. DAB-treated paraffin sections of colorectal cancer were immersed in xylene solution for 15 minutes to dissolve the mounting medium, and then the coverslip was removed. The sections were then incubated in 0.5% potassium permanganate solution at room temperature for 2 minutes, rinsed with deionized water, immersed in 2% oxalic acid solution for 1 minute, rinsed under running water for 5 minutes, dried, and then mounted with a mounting medium, covered with a coverslip, and observed under a microscope.

[0058] The inspection results are as follows Figure 6 As shown.

[0059] The results showed that the conventional potassium permanganate-oxalic acid method could remove most of the DAB color, leaving a light yellow residue, but the tissue morphology was severely damaged.

[0060] Comparative Example 2 Colorectal cancer tissue stained with CK pan was selected as the destained section using standard 3% hydrogen peroxide-methanol solution. DAB-treated paraffin sections of colorectal cancer were immersed in xylene solution for 15 minutes to dissolve the mounting medium, and then the coverslip was removed. The sections were then immersed in a 3% hydrogen peroxide-methanol solution, where hydrogen peroxide and methanol were mixed at a 1:1 volume ratio. Incubation was performed at room temperature for 10 minutes, followed by rinsing with running water for 5 minutes. After drying, the sections were covered with mounting medium and covered with coverslips for observation under a microscope.

[0061] Test results as follows Figure 7 As shown.

[0062] The results showed that the conventional 3% hydrogen peroxide-methanol method was not effective in decolorizing the sections after DAB staining.

[0063] Example 6 Colon cancer sections stained with CK pan were destained using a destaining solution containing 20% ​​1-ethyl-3-methylimidazolium sulfate and 0.1% neutral protease (dispersant) and a destaining solution containing 0.5% potassium permanganate-2% oxalic acid. The sections were then counterstained with CK8 / 18, and the staining intensity and tissue integrity after counterstaining were compared.

[0064] The steps are as follows: 1. Place two DAB-developed paraffin sections of colon cancer in xylene solution for 15 minutes to dissolve the mounting medium, then remove the coverslip. 2. Option 1: Perform the same steps as in Comparative Example 1 on a slice; Option 2: Decolorize a slice by using a decolorizing solution containing 20% ​​1-ethyl-3-methylimidazolium sulfate and 0.1% neutral protease (dispersant) according to the steps in Example 1; 3. After decolorization, add 150 µL of CK8 / 18 antibody and incubate at room temperature for 30 minutes, then wash with PBST 3 times, 1 minute each time; 4. Add the polymeric secondary antibody and incubate at room temperature for 30 minutes, then wash three times with PBST for 1 minute each time; 5. Add DAB and develop color for 3 minutes, then rinse with running water for 1 minute. 6. Counterstain with hematoxylin for 5 minutes, rinse with running water, and then blue with PBST for 10 seconds; 7. Dehydrate with graded alcohols, clear with xylene, dry, mount, and observe the results under a microscope.

[0065] Test results as follows Figure 8 As shown.

[0066] The results showed that the strength of Scheme 2, which used the method described in this application for decolorization and then re-dyeing, was much higher than that of Scheme 1, which used potassium permanganate-oxalic acid decolorization. Moreover, the tissue structure was more intact and there was no tissue damage.

[0067] The method described in this application can better preserve the antigen activity in the tissue and can be used for decolorization methods that require multiple stainings on the same tissue section.

[0068] All documents mentioned in this invention are incorporated herein by reference as if each document were individually incorporated by reference. Furthermore, it should be understood that after reading the foregoing teachings of this invention, those skilled in the art can make various alterations or modifications to this invention, and these equivalent forms also fall within the scope defined by this application.

Claims

1. A DAB immunohistochemical staining destaining solution, characterized in that, The decolorizing solution comprises a functionalized ionic liquid with a volume concentration of 10%-40% and a co-solvent of 60%-90%. The functionalized ionic liquid is a carboxyl-functionalized imidazole ionic liquid and / or a hydroxypyridine ionic liquid; The co-solvent is a glycerol solution.

2. The DAB immunohistochemical staining destaining solution according to claim 1, characterized in that, The functionalized ionic liquid is one or a mixture of two of 1-ethyl-3-methylimidazolium sulfate and N-ethylpyridine tetrafluoroborate.

3. The DAB immunohistochemical staining destaining solution according to claim 1, characterized in that, The mass concentration of glycerol in the co-solvent is 5%.

4. The DAB immunohistochemical staining destaining solution according to claim 1, characterized in that, The pH of the decolorizing solution is 6.8-7.

5.

5. A DAB immunohistochemical staining and destaining solution according to any one of claims 1-4, characterized in that, The decolorizing solution also includes an enzymatic hydrolysis component with a volume concentration of 0.05%-0.2%.

6. The DAB immunohistochemical staining destaining solution according to claim 5, characterized in that, The enzymatic hydrolysate is a neutral protease.

7. A method for DAB immunohistochemical staining and destaining using the destaining solution according to any one of claims 1-6, characterized in that, Includes the following steps: S1. Take the pathological sections after DAB staining and demount them in xylene solution; S2. The tissue area is completely covered with the decolorizing solution according to any one of claims 1-6, incubated at room temperature, and observed under a microscope until the DAB brownish-yellow color completely disappears. S3. Rinse the slices with deionized water to completely remove the DAB color.

8. The method according to claim 7, characterized in that, In step S1, the slides are desealed in xylene solution for 10-20 minutes.

9. The method according to claim 7, characterized in that, In step S2, the greenhouse incubation time is 5-10 minutes.

10. The method according to claim 7, characterized in that, In step S3, the slides are rinsed with deionized water 2-3 times, each time for at least 1 minute.

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