CN117929065A - Novel universal immunohistochemical antigen retrieval liquid and preparation method thereof - Google Patents
Novel universal immunohistochemical antigen retrieval liquid and preparation method thereof Download PDFInfo
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- CN117929065A CN117929065A CN202410128215.5A CN202410128215A CN117929065A CN 117929065 A CN117929065 A CN 117929065A CN 202410128215 A CN202410128215 A CN 202410128215A CN 117929065 A CN117929065 A CN 117929065A
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- Investigating Or Analysing Biological Materials (AREA)
Abstract
The invention discloses a novel general immunohistochemical antigen retrieval liquid and a preparation method thereof, wherein each 1000mL of the antigen retrieval liquid comprises the following components: tris1.8-1.9g, EGTA0.35-0.4g, AMP480-520 muL and the balance of water. The AMP in the antigen retrieval liquid component can enable the antigen retrieval to be more thorough; AMP has the function of protecting the integrity of protein and cell membrane, and can make the cell morphology of immunohistochemical section result intact; AMP has the effect of stabilizing the pH of a solution, and does not need to be readjusted when preparing antigen retrieval liquid; the EGTA has strong specificity, and has better repairing effect when being used for repairing antigens; the antigen retrieval liquid is used for immunohistochemical staining, and the background is clean while the strength of a section staining result is better.
Description
Technical Field
The invention relates to the technical field of immunohistochemistry, in particular to novel universal immunohistochemical antigen restoration liquid and a preparation method thereof.
Background
In the technical field of pathological diagnosis, the immunohistochemical technique has important auxiliary diagnosis effect and is valued in clinical application. The immunohistochemical technology uses the principle of high specific binding between antibody and antigen, uses the antibody to specifically bind the target protein in tissue or cell as antigen, and then displays the binding position of the antibody by chemical method to achieve the research of qualitative, positioning or quantification of unknown antigen in tissue or cell. Before immunohistochemistry, some reagents (such as paraformaldehyde) are often used for treating and fixing fresh tissues or cells, and formaldehyde in the tissues or cells are crosslinked with protein in the tissue fixing process to form aldehyde crosslinked protein, and the compound seals antigen so that the antigen cannot be expressed. In order to better expose the antigen during immunohistochemistry, it is well shown that the world-accepted method is that antigen retrieval is necessary.
The antigen retrieval has extremely important effect in the immunohistochemical process, is an indispensable step in the immunohistochemical technology, and can obtain accurate and reliable experimental results by recovering the activity of the antigen, and the quality of the antigen retrieval results directly determines the dyeing quality of the immunohistochemical results. There are various methods for antigen retrieval, and some of these methods are performed according to the requirements of antibodies, and others are performed according to the expression level of an antigen. The enzyme digestion process is not the main method, and the heat repair is more common, so the repair effect is most effective, structural damage and other side effects are less, and the operation is simpler and more convenient.
The basic principle of thermal remediation is: the protein is placed in an aqueous environment and heating accelerates the proteolytic process, thereby opening the chemical linkage formed by the immobilized cross-links, exposing the previously masked antigenic determinants. There are many factors determining the healing effect of thermal healing, such as the type of healing liquid, pH of the healing liquid, and the healing temperature. The most commonly used antigen retrieval solutions are of two types: sodium citrate buffer (ph=6) and EDTA buffer (ph=8 or 9). The sodium citrate buffer solution can be suitable for most antibodies, but the overall repair effect is poor due to the lower pH value of the sodium citrate buffer solution; EDTA repair liquid has stronger liquid repair effect, but positive enhancement can also improve non-specific background, and when preparing EDTA repair liquid, usually need to adjust pH with sodium hydroxide, sodium hydroxide is strong base for when adjusting pH, the use amount is not well controlled, and if careless when using will cause injury to laboratory staff, and corrode laboratory etc..
Disclosure of Invention
The invention aims to solve the technical problems of poor citric acid repairing effect, excessive EDTA repairing background and the like by providing a novel universal immunohistochemical antigen repairing liquid and a manufacturing method thereof.
The technical problems to be solved by the invention are realized by the following technical scheme:
The novel general immunohistochemical antigen retrieval liquid comprises the following components in every 1000 mL: tris 1.8-1.9 g, ethylene glycol bis alpha-aminoethyl ether tetraacetic acid (EGTA) 0.35-0.4 g, aminomethylpropanol (AMP) 480-520 mu L, and the balance water.
Preferably, each 1000mL of the novel universal immunohistochemical antigen retrieval liquid comprises: tris1.85g, ethylene glycol bis [ alpha ] -aminoethylether tetraacetic acid (EGTA) 0.38g, aminomethylpropanol (AMP) 500. Mu.L, the balance being water.
Preferably, the water is deionized water.
The preparation method of the novel universal immunohistochemical antigen retrieval liquid comprises the following steps:
(1) Weighing Tris according to the formula amount, and completely dissolving the Tris by 500mL of distilled water;
(2) Weighing EGTA according to the formula amount, and completely dissolving the EGTA in the step (1);
(3) AMP is measured and dissolved in the step (2) according to the formula amount;
(4) Finally, the volume was fixed with water to 1000mL.
Preferably, the water is deionized water.
The application of the novel universal immunohistochemical antigen retrieval liquid in the field of tissue immunohistochemistry or the field of cell immunohistochemistry.
An application of novel universal immunohistochemical antigen retrieval liquid in immunohistochemical staining.
The technical scheme of the invention has the following beneficial effects:
(1) The AMP in the antigen retrieval liquid component can enable the antigen retrieval to be more thorough;
(2) The AMP in the antigen retrieval liquid component has the function of protecting the integrity of protein and cell membranes, and can ensure that the cell morphology of an immunohistochemical section result is intact;
(3) The AMP in the antigen retrieval liquid component provided by the invention has the function of stabilizing the pH of the solution, and the pH is not required to be regulated when the antigen retrieval liquid is prepared;
(4) The EGTA in the antigen retrieval liquid component provided by the invention has strong specificity, and has better retrieval effect when being used for antigen retrieval;
(5) The antigen retrieval liquid provided by the invention is used for immunohistochemical staining, and the slice staining result has better strength and clean background.
Drawings
The accompanying drawings, which are incorporated in and constitute a part of this specification, illustrate embodiments of the invention and together with the description, serve to explain the principles of the invention.
Fig. 1 is a graph showing the dyeing effects of the examples and comparative examples of the present invention (the left graph is the example, and the right graph is the comparative example).
FIG. 2 is a comparative plot of cell morphology and staining background for examples of the invention versus comparative examples (left panel is example, right panel is comparative example).
Detailed Description
Various exemplary embodiments of the present invention will now be described in detail. It should be noted that: the relative arrangement of the components and steps, numerical expressions and numerical values set forth in these embodiments do not limit the scope of the present invention unless it is specifically stated otherwise.
Example 1
The novel general immunohistochemical antigen retrieval liquid comprises the following components in every 1000mL of the novel general immunohistochemical antigen retrieval liquid: tris 1.8g, EGTA0.35g, AMP480. Mu.L, the balance deionized water.
The preparation method comprises the following steps:
(1) Weighing Tris according to the formula amount, and completely dissolving the Tris by 500mL of distilled water;
(2) Weighing EGTA according to the formula amount, and completely dissolving the EGTA in the step (1);
(3) Using a pipette to measure and measure the AMP according to the formula amount to be dissolved in the step (2);
(4) Finally, the volume is fixed to 1000mL by water, and the recombinant DNA can be used for repairing the immunohistochemical antigen.
Example 2
The novel general immunohistochemical antigen retrieval liquid comprises the following components in every 1000mL of the novel general immunohistochemical antigen retrieval liquid: tris 1.9g, EGTA0.4g, AMP520. Mu.L, the balance deionized water.
The preparation method is the same as in example 1.
Example 3
The novel general immunohistochemical antigen retrieval liquid comprises the following components in every 1000 mL: tris 1.85g,EGTA0.38g,AMP 500. Mu.L, the balance water.
The preparation method is the same as in example 1.
Application example 1 immunohistochemical antigen retrieval assay
Tissue staining: tonsil, intestinal, adrenal cortex and mammary gland tissues were selected for immunohistochemical staining, and primary antibodies were selected for testing as bcl-6, MLH-1, inhibin α, ki67, PMS2, CD56, CD68 and PCNA. After a series of pretreatment, the tissues were paraffin-embedded and sliced to a slice thickness of 3 μm, and baked according to the requirements of the laboratory baking, immunohistochemical staining test was performed using the novel antigen retrieval solutions of examples 1-3.
Comparative example 1
The common conventional EDTA antigen repair liquid is used for testing, and the selected tissues, the test primary antibodies and the test process are consistent with the application examples.
The results are shown in FIGS. 1 and 2. The example in the figure is the result of example 3. As shown in fig. 1 and 2, the staining results of the embodiment 3 and the comparative example 1 show that the embodiment of the application can make the staining intensity of the section better, the cell morphology perfect, and the background clean.
The invention provides a novel antigen retrieval liquid capable of eliminating formaldehyde protein cross-linked matters formed between a fixation liquid and tissues or cells based on the principle of tissue or cell fixation. The invention selects nontoxic amine chemical substance Aminomethylpropanol (AMP) for preparing novel repair liquid, which can be used for protecting protein and cell membrane integrity, promoting antigen stability and visualization, and achieving the purpose of improving cell morphology and tissue background. AMP also has the effect of stabilizing the pH of the solution, and no pH adjustment is required in the formulation of AMP antigen retrieval solutions.
Although the present invention has been described with reference to the above embodiments, it should be understood that the present invention is not limited thereto, and that various changes and modifications may be made by those skilled in the art without departing from the spirit and scope of the present invention, and the scope of the present invention is defined by the appended claims and their equivalents.
Claims (7)
1. The novel general immunohistochemical antigen retrieval liquid is characterized in that each 1000mL of the novel general immunohistochemical antigen retrieval liquid comprises: tris 1.8-1.9 g, ethylene glycol bis alpha-aminoethyl ether tetraacetic acid (EGTA) 0.35-0.4 g, aminomethylpropanol (AMP) 480-520 mu L, and the balance water.
2. The novel universal immunohistochemical antigen retrieval solution as recited in claim 1, wherein each 1000mL of the novel universal immunohistochemical antigen retrieval solution comprises: tris 1.85g, ethylene glycol bis [ alpha ] -aminoethylether tetraacetic acid (EGTA) 0.38g, aminomethylpropanol (AMP) 500. Mu.L, the balance being water.
3. The novel universal immunohistochemical antigen retrieval solution as recited in claim 1, wherein the water is deionized water.
4. A method for preparing a novel universal immunohistochemical antigen retrieval solution according to any one of claims 1 to 3, comprising the steps of:
(1) Weighing Tris according to the formula amount, and completely dissolving the Tris by 500mL of distilled water;
(2) Weighing EGTA according to the formula amount, and completely dissolving the EGTA in the step (1);
(3) AMP is measured and dissolved in the step (2) according to the formula amount;
(4) Finally, the volume was fixed with water to 1000mL.
5. The method for preparing a novel universal immunohistochemical antigen retrieval solution as recited in claim 4, wherein the water is deionized water.
6. Use of a novel universal immunohistochemical antigen retrieval solution according to any one of claims 1 to 3 in the field of tissue immunohistochemistry or in the field of cytoimmunohistochemistry.
7. Use of a novel universal immunohistochemical antigen retrieval solution according to any one of claims 1-3 in immunohistochemical staining.
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| Application Number | Priority Date | Filing Date | Title |
|---|---|---|---|
| CN202410128215.5A CN117929065A (en) | 2024-01-30 | 2024-01-30 | Novel universal immunohistochemical antigen retrieval liquid and preparation method thereof |
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| Application Number | Priority Date | Filing Date | Title |
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| CN202410128215.5A CN117929065A (en) | 2024-01-30 | 2024-01-30 | Novel universal immunohistochemical antigen retrieval liquid and preparation method thereof |
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| Publication Number | Publication Date |
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| CN117929065A true CN117929065A (en) | 2024-04-26 |
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| CN202410128215.5A Withdrawn CN117929065A (en) | 2024-01-30 | 2024-01-30 | Novel universal immunohistochemical antigen retrieval liquid and preparation method thereof |
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| Country | Link |
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| CN (1) | CN117929065A (en) |
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- 2024-01-30 CN CN202410128215.5A patent/CN117929065A/en not_active Withdrawn
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Application publication date: 20240426 |