CN110567785B

CN110567785B - Staining kit and method of use

Info

Publication number: CN110567785B
Application number: CN201910804447.7A
Authority: CN
Inventors: 李立科; 蒋小龙
Original assignee: Maccura Biotechnology Co ltd
Current assignee: Maccura Biotechnology Co ltd
Priority date: 2019-08-28
Filing date: 2019-08-28
Publication date: 2022-04-08
Anticipated expiration: 2039-08-28
Also published as: CN110567785A

Abstract

The invention relates to a staining kit, which comprises a first reagent containing epigallocatechin gallate or derivatives thereof and DAB chromogen and can improve the stability of DAB staining working solution. The invention also relates to a method for using the kit.

Description

Staining kit and method of use

Technical Field

The invention relates to the fields of pathology and molecular biology, in particular to a DAB staining kit.

Background

Immunohistochemistry, abbreviated as immunohistochemistry, is an immunological method in which an antibody is labeled with a tracer to search for an antigen in a tissue cell, i.e., a chemical component in a tissue cell, and thus a color change is also used to determine the chemical component in the tissue cell. Occasionally, antibodies in tissues are also detected by means of labeled antigens. The method has the advantages of high sensitivity, strong specificity, qualitative and quantitative properties and the like, and is widely used for detecting tissue paraffin sections, tissue frozen sections, cell smears, cell prints and cultured cells.

In situ hybridization refers to the process of hybridizing specifically labeled nucleic acids of known sequence as probes to nucleic acids in cells or tissue sections to precisely quantify the location of a particular nucleic acid. In situ hybridization can be performed on a cell or tissue specimen. The method has high sensitivity and specificity, and can further discuss the functional expression and the regulation mechanism of the cell from the molecular level. Has become an important means for the research of cell biology and molecular biology at present.

Western blotting, also known as Western Blot, is a protein detection technique in which the total protein of cells or tissues after electrophoretic separation is transferred from a gel to a solid support NC membrane or PVDF membrane, and then a specific antigen is detected using a labeled specific antibody, and has been widely used in many fields such as gene expression studies at protein level, antibody activity detection, and early diagnosis of diseases.

In the immunohistochemistry, in situ hybridization and Western Blot methods, after the reaction, a colorless substance is converted to a certain color through a certain chemical reaction so as to be able to be observed under a microscope. According to different labeled tracers, the corresponding color development system is suitable. As labeled tracer, horseradish peroxidase (HRP) is currently commonly used, which generally uses DAB as a hydrogen donor, H₂O₂As a substrate, the final product of antigen-antibody binding is finally made tan or brownish. HRP-specific substrate is H₂O₂In the decomposition of H₂O₂In the process, HRP and H₂O₂Forming a primary complex, the reaction not continuing without an electron donor; when the electron donor is present, the reaction forms a second complex at a certain rate, after which the HRPCatalysis H₂O₂The intermediate product is produced into water fast, the enzyme is reduced, the electron donor is oxidized, polymerized and oxidized to form indoleamine polymer, which is further oxidized and cyclized to form phenyl ethyl hydrazine polymer. The final product can be directly observed under a light microscope or can be subjected to OsO₄After treatment, the electron density of the reaction product is increased for electron microscope observation.

At present, the DAB staining solution used traditionally has the main disadvantages that: because the DAB coloring agent has poor stability, the DAB coloring agent is easy to gradually lose hydrogen supply capability in a solution, so that the final dyeing fails, and the DAB coloring agent needs to be prepared for use.

Accordingly, there is a strong need in the art to improve the stability of DAB staining solutions.

Disclosure of Invention

Currently, the problem of improving the stability of DAB colorants has attracted attention. In the process of solving the problem, the inventors tried to add various stabilizers, such as propylene glycol, glycerol, edta.2na, trehalose or casein, into the DAB solution, and the results show that the stabilizers have a certain improvement effect on the stability of the DAB staining solution, but the effect is still not ideal (as illustrated in example 2), mainly reflected in poor stability in the medium and long periods after three days. In addition, the use of vitamin C with reducing action as a stabilizer was attempted, but the results showed that DAB stain did not stain antigens in color after addition of vitamin C (as illustrated in example 3). However, the inventors have unexpectedly found in further studies that: when epigallocatechin gallate, which also has reducibility, is used as a stabilizer, a good stabilizing effect can be obtained, and the present invention has been completed. Without wishing to be bound by theory, we speculate that this may be due to the simultaneous antibacterial effect of epigallocatechin gallate.

Accordingly, in a first aspect, the present invention provides a kit capable of preserving DAB for a long period of time, the kit comprising:

a first reagent comprising epigallocatechin gallate or a derivative thereof and DAB chromogen.

In the kit of the present invention, epigallocatechin gallate can effectively improve the stability of DAB chromogen without affecting the determination of DAB staining on positive result, so that the prepared DAB staining working solution can be stored for a longer time at normal temperature. That is, after the kit of the present invention is used, the prepared DAB staining solution can be used immediately or after being stored for several days, and both of them can ensure the sensitivity and reliability of detection. In this way, the operator no longer needs to ensure the dyeing effect by means of the existing preparation, which on the one hand saves reagents, reduces the detection costs and saves the detection time, and on the other hand, the invention avoids the environmental pollution caused by the excess liquid due to the potential carcinogenicity of DAB.

In a preferred embodiment, the concentration of epigallocatechin gallate or a derivative thereof in the first reagent is from about 25. mu.M to about 10mM, such as from about 50. mu.M to about 5 mM.

Compared with common stabilizers such as propylene glycol, glycerol, EDTA.2Na, trehalose or casein, the stability of the DAB dyeing working solution after 3 or 7 days of storage is further improved at such concentrations; in addition, the dyeing effect of the dyeing working solution after being stored for 7 days is improved to a certain extent, so that the detection cost is further reduced, the pollution is avoided, and the clinical use is facilitated.

In a further preferred embodiment, the concentration of epigallocatechin gallate or a derivative thereof in the first reagent is from about 0.25mM to about 10mM, such as from about 0.5mM to about 5 mM.

By selecting such a concentration range, the storage time of the DAB staining solution was further prolonged and the staining effect was improved so that the DAB staining solution could be used as it was after 7 days of preparation.

It will be appreciated that the first reagent of the present invention may be provided as part of a DAB staining kit which provides a DAB chromogen. Before dyeing, the first reagent of the invention can be mixed with DAB substrate and DAB buffer solution to prepare DAB dyeing working solution.

In the present invention, the terms "DAB chromogen" and "DAB color source" are used interchangeably and refer to a DAB developer comprising 3, 3' -diaminobenzidine or salts, hydrates and solvates thereof. In particular embodiments, the DAB chromogen may be, for example, 3 '-diaminobenzidine, 3' -diaminobenzidine tetrahydrochloride, 3 'diaminobenzidine tetrahydrochloride hydrate, 3' diaminobenzidine carbon tetrachloride, or a combination thereof. In the first reagent, the DAB chromogen may be present in a suitable solvent (water soluble or lipid soluble) at conventional concentrations, with exemplary concentrations ranging from about 5 to about 80 mM.

In the present invention, Epigallocatechin gallate (EGCG) is abbreviated as EGCG and its CAS number is 989-51-5. In the present invention, the derivative of epigallocatechin gallate means a product obtained by subjecting epigallocatechin gallate to methylation modification, acylation modification, esterification modification, glycoside modification or the like.

In the present invention, the DAB substrate may be a substrate required in the HRP catalyzed reaction, and an exemplary DAB substrate may be hydrogen peroxide (30%), which may be present, for example, in an amount of 0.1% to 5%.

As for DAB buffer, the present invention is not particularly limited as long as it provides a desired environment for DAB staining, and exemplary DAB buffers may be a buffer containing 50 to 200nM Tris, 20 to 100mM imidazole and 1 to 20mM Brij-35, Tris-HCl buffer, glycine-HCl buffer or phosphate buffer (e.g., disodium hydrogen phosphate-sodium dihydrogen phosphate buffer, disodium hydrogen phosphate-potassium dihydrogen phosphate buffer, potassium dihydrogen phosphate-sodium hydroxide buffer), but the present invention is not limited thereto. In particular embodiments, the pH of the buffer may be, for example, 7.6 ± 0.3.

In case of use as DAB staining kit, the DAB substrate and DAB buffer may be present alone or in combination. For example, in one embodiment, a kit of the invention may comprise: a first reagent containing epigallocatechin gallate or a derivative thereof and DAB chromogen; a second reagent containing a DAB substrate; and a third reagent containing DAB buffer. In another embodiment, the kit of the present invention may comprise: a first reagent containing epigallocatechin gallate or a derivative thereof and DAB chromogen; a second reagent comprising a DAB substrate and a DAB buffer.

In the present invention, the expressions "first", "second" and "third", etc. are used for descriptive purposes only to distinguish defined objects, and not to define an order or primary or secondary in any way.

It will be appreciated that the kit of the invention may also include other components, for example, hematoxylin staining solution and the like, when it is desired to counterstain the cells. For another example, in the case of use in immunohistochemical analysis, the kit may further comprise secondary antibodies, blocking solutions, endogenous peroxidase blockers, and the like.

In one embodiment, the kit of the invention is used for immunohistochemical analysis.

In another embodiment, the kit of the invention is used in an in situ hybridization assay.

In yet another embodiment, the kit of the invention is used for western blot analysis.

In a second aspect, the present invention provides a method for improving the stability of a DAB staining solution, comprising contacting said DAB chromogen with epigallocatechin gallate or a derivative thereof.

In one embodiment, the present invention provides a method for improving the stability of a DAB dyeing working solution, comprising adding epigallocatechin gallate or a derivative thereof to the DAB dyeing working solution.

In a preferred embodiment, the concentration of epigallocatechin gallate or a derivative thereof in the DAB staining solution is about 1 μ M to 400 μ M, such as about 1.5 μ M to 300 μ M, and such as about 2 μ M to 200 μ M.

In a further preferred embodiment, in the DAB staining working solution, the concentration of epigallocatechin gallate or a derivative thereof is about 10 μ M to 400 μ M, such as about 15 μ M to 300 μ M, and such as about 20 μ M to 200 μ M.

In a third aspect, the present invention provides a dyeing method comprising:

mixing a first reagent containing epigallocatechin gallate or derivatives thereof and DAB chromogen with a DAB substrate and a DAB buffer solution to prepare a working solution;

adding the prepared working solution into a sample to be dyed and incubating; and

the color development was terminated.

In one embodiment, the method further comprises the step of counterstaining with hematoxylin staining solution.

In a preferred embodiment, the concentration of epigallocatechin gallate or a derivative thereof in the first reagent is from about 25 μ M to about 10mM, such as from about 50 μ M to about 5 mM.

In the method of the invention, the working fluid is allowed to stand for 2 days or more, for example 3 days or more, and for example 7 days or more, before being added to the sample to be stained.

Detailed Description

The present invention will be described in detail below with reference to specific embodiments and examples, and the advantages and various effects of the present invention will be more clearly apparent therefrom. It will be understood by those skilled in the art that these specific embodiments and examples are for the purpose of illustrating the invention and are not to be construed as limiting the invention.

Dyeing method

a) Dewaxing and hydrating:

dipping the paraffin section in dimethylbenzene for 5 minutes and multiplying by 2 times; after removing the redundant liquid, placing the mixture in absolute ethyl alcohol, and soaking for 3 minutes and 2 times; removing the excessive liquid, and sequentially soaking in 95%, 85% and 75% ethanol for 3 min each time; washing with tap water for 1 minute; wash with PBS solution 3 min × 3 times.

b) Antigen retrieval: antigen retrieval was performed using immunohistochemical antigen retrieval buffer (michael organism, cat # PD3101014, lot # 0519031, or michael organism, cat # PD3101015, lot # 0419011) according to the manufacturer's instructions.

c) Endogenous peroxidase blockade:

after the antigen is repaired, the naturally cooled section is used for circling the tissue area to be detected on the slide by an oil pen, and PBS solution is washed for 3 minutes and 3 times; removing the redundant liquid, dripping an endogenous peroxidase blocking agent into the area defined by the oil pen, and incubating for 10 minutes at room temperature in a dark place; the PBS solution was washed 3 min X3 times.

d) Primary antibody incubation:

adding a primary antibody (a mouse anti-human p40 monoclonal antibody, China fir gold bridge, cargo number ZM-4072, batch number 19021108, or a rabbit anti-human p40 polyclonal antibody, Fuzhou maixin, cargo number RMA-0815, batch number 1902280815C1) working solution dropwise into the area defined by the oil pen, and incubating (the incubation conditions are operated according to the primary antibody specification); after incubation, the PBS solution was washed 3 min x 3 times.

e) And (3) secondary antibody incubation:

excess liquid was removed, an enzyme-labeled goat anti-mouse/rabbit IgG polymer was added dropwise, incubated at 37 ℃ for 30 minutes, and washed with PBS solution 3 minutes × 3 times.

f) DAB color development:

removing redundant liquid, dropwise adding the prepared DAB dyeing working solution, incubating for 2-5 minutes at room temperature, and washing with tap water to stop color development.

g) Counterdyeing:

counterstaining with hematoxylin staining solution until cell nucleus is light blue, and washing with tap water to turn blue.

h) Dehydration and transparency:

placing the mixture in 75%, 85%, 95% and absolute ethyl alcohol in sequence for dehydration for 3 minutes each time; clear 5 min x 2 times in xylene.

i) Sealing: sealing with neutral gum, and reading with a biological microscope.

Example 1

The formulations of the reagents were made as in table 1 below:

TABLE 1

DAB buffer solution, DAB substrate (dissolved in 0.02M phosphate buffer solution) and DAB chromogen (dissolved in 0.02M phosphate buffer solution) are prepared into DAB dyeing working solution according to the proportion of 1ml:40 muL, and the DAB dyeing working solution is respectively stored for 0 day, 3 days and 7 days at room temperature after preparation, then dyeing is carried out according to the dyeing method, and the dyeing effect of different storage days is observed under a microscope and compared, and the results are shown in the following table 2.

TABLE 2

From the results under the microscope, it was found that the staining solution was able to stain the antigen in the tissue cells in a brown-yellow color, showing strong positive and no background color, when left at room temperature for 0 day. After hematoxylin counterstaining, the cell nucleus is bluish purple, and the whole tissue is stained brightly with bright contrast. Meanwhile, the DAB dyeing working solution prepared freshly is light yellow without precipitates. After being placed at room temperature for 3 days, compared with 0 day, the positive signals are weakened to a certain extent, and part of positive cells are not obviously colored and show weak positive. Meanwhile, the color of the DAB dyeing working solution is changed from light yellow to brown yellow, and a small part of particles or flocculent precipitates exist. After being placed at room temperature for 7 days, compared with 0 day, the positive signal is obviously weakened, and part of positive cells become light or not colored and become weak positive or negative. Meanwhile, the color of the DAB dyeing working solution is changed from brown to brown, and the DAB dyeing working solution has granular or flocculent precipitates.

Example 2

On the basis of example 1, different protective agents are respectively added into the DAB chromogen solution, and the rest components, preparation steps and test process are unchanged, and the results are shown in the following table 3.

TABLE 3

As is clear from table 3, when 1% propylene glycol, 1% glycerol, 0.3% edta.2na, 1% trehalose, or 1% casein was added as a stabilizer, the staining solution was able to stain the antigen in the tissue cells in a brown-yellow color after being left at room temperature for 0 day, and showed strong positive and no background color. After hematoxylin counterstaining, the cell nucleus is bluish purple, and the whole tissue is bright-colored in staining and bright in contrast. Meanwhile, the DAB dyeing working solution prepared freshly is light yellow without precipitates. After 3 days at room temperature, the decrease in the intensity of the positive signal was weak positive compared with 0 day. Meanwhile, the color of the DAB dyeing working solution is changed from light yellow to brown yellow, and a small part of particles or flocculent precipitates exist. After being placed at room temperature for 7 days, compared with 0 day, the positive signal is obviously weakened, a few positive cells are not colored, and the cells become weak positive or negative. Meanwhile, the color of the DAB dyeing working solution is changed from brown to brown, and the DAB dyeing working solution has granular or flocculent precipitates.

That is, the addition of propylene glycol, glycerol, edta.2na, trehalose and casein as stabilizers can protect DAB to some extent, maintain its ability to provide electrons, and thus improve the stability of DAB staining solution. However, the protective effect of these stabilizers in the middle and later stages of the dyeing working solution is obviously weakened, which results in the dyeing failure and precipitation of the dyeing working solution, which is not favorable for the continuity of clinical pathological diagnosis operation, and also causes the waste of redundant reagents and increases the burden of medical resources.

Example 3

On the basis of example 1, different reducing agents were added to the DAB chromogen solution as stabilizers, and the remaining components, formulation steps and test procedures were unchanged, with the results shown in table 4 below.

TABLE 4

As can be seen from table 4, when vitamin C was added as a stabilizer, the DAB staining solution became pale yellow, orange yellow and orange yellow in color after being left at room temperature for 0, 3 and 7 days, and none of them could stain the antigen in the tissue cells and showed negative results. With epigallocatechin gallate as stabilizer, the staining solution can stain antigen in tissue cell to brown yellow, showing strong positive and no background color after being placed at room temperature for 0 day. After hematoxylin counterstaining, the cell nucleus is bluish purple, and the whole tissue is bright-colored in staining and bright in contrast. Meanwhile, the DAB dyeing working solution prepared freshly is light yellow without precipitates. After standing at room temperature for 3 days, the intensity of the positive signal was unchanged from 0 day, and the signal was strongly positive. Meanwhile, the DAB working solution is slightly deepened but not obvious in color and free of precipitate. After 7 days at room temperature, the positive signal was somewhat attenuated but not significant compared to day 0. Meanwhile, the color of the DAB dyeing working solution is changed from light yellow to brown yellow without precipitation.

Example 4

On the basis of example 1, epigallocatechin gallate with different concentrations is added into the DAB chromogen solution, and the rest components, preparation steps and test process are unchanged, and the results are shown in the following table 5.

TABLE 5

As is clear from Table 5, when epigallocatechin gallate (0.5 mM) was added as a stabilizer in the same manner as when the amount was 5mM, the staining solution was able to stain the antigen in the tissue cells in a brown-yellow color, showing strong positive and no background color after 0 day of standing at room temperature. After hematoxylin counterstaining, the cell nucleus is bluish purple, and the whole tissue is bright-colored in staining and bright in contrast. Meanwhile, the DAB dyeing working solution prepared freshly is light yellow without precipitates. After standing at room temperature for 3 days, the intensity of the positive signal was unchanged from 0 day, and the signal was strongly positive. Meanwhile, the DAB working solution is slightly deepened but not obvious in color and free of precipitate. After 7 days at room temperature, the positive signal was somewhat attenuated but not significant compared to day 0. Meanwhile, the color of the DAB dyeing working solution is changed from light yellow to brown yellow without precipitation. With the addition of 50 μ M epigallocatechin gallate as a stabilizer, the staining solution was able to stain the antigen in the tissue cells to a tan color, showing strong positive, with no background color after 0 days at room temperature. After hematoxylin counterstaining, the cell nucleus is bluish purple, and the whole tissue is bright-colored in staining and bright in contrast. Meanwhile, the freshly prepared DAB dyeing working solution is light yellow and has no precipitate; after being placed at room temperature for 3 days, the positive signals are weakened to a certain extent compared with 0 day, and partial positive cells are lightened in staining and are expressed as weak positive. Meanwhile, the color of the DAB working solution is slightly deepened, and no precipitate exists. After being placed at room temperature for 7 days, compared with 0 day, the intensity of the positive signal is obviously weakened, part of positive cells are not obviously colored, and the cells become weak positive. Meanwhile, the color of the DAB working solution is changed from light yellow to brown without precipitation.

Claims

1. A kit, comprising:

2. The kit according to claim 1, wherein the concentration of epigallocatechin gallate or a derivative thereof is from 25 μ M to 10 mM.

3. The kit according to claim 2, wherein the concentration of epigallocatechin gallate or a derivative thereof is 50 μ M to 5 mM.

4. The kit according to claim 2, wherein the concentration of epigallocatechin gallate or a derivative thereof is from 0.25mM to 10 mM.

5. The kit according to claim 2, wherein the concentration of epigallocatechin gallate or a derivative thereof is from 0.5mM to 5 mM.

6. The kit of any one of claims 1 to 5, further comprising a second reagent comprising a DAB substrate and a third reagent comprising a DAB buffer.

7. The kit of any one of claims 1 to 5, further comprising a second reagent comprising a DAB substrate and a DAB buffer.

8. The kit of any one of claims 1 to 5, wherein the kit is an immunohistochemical kit.

9. A method for improving the stability of DAB dyeing working solution comprises the step of contacting DAB chromogen with epigallocatechin gallate or derivatives thereof.

10. The method of claim 9, wherein the concentration of epigallocatechin gallate or a derivative thereof in the DAB staining solution is from 1 μ Μ to 400 μ Μ.

11. The method of claim 10, wherein the concentration of epigallocatechin gallate or a derivative thereof in the DAB staining solution is 2 μ Μ to 200 μ Μ.

12. The method of claim 10, wherein the concentration of epigallocatechin gallate or a derivative thereof in the DAB staining solution is from 10 μ Μ to 400 μ Μ.

13. The method of claim 10, wherein the concentration of epigallocatechin gallate or a derivative thereof in the DAB staining solution is 20 μ Μ to 200 μ Μ.

14. A method of dyeing comprising:

preparing a first reagent containing epigallocatechin gallate or derivatives thereof and DAB chromogen, a DAB substrate and a DAB buffer solution into a working solution;

adding the working solution to a sample to be dyed and incubating; and

the color development was terminated.

15. The method of claim 14, wherein the concentration of epigallocatechin gallate or a derivative thereof in the first reagent is from 25 μ Μ to 10 mM.

16. The method of claim 15, wherein the concentration of epigallocatechin gallate or a derivative thereof in the first reagent is between 50 μ Μ and 5 mM.

17. The method of claim 15, wherein the concentration of epigallocatechin gallate or a derivative thereof in the first reagent is from 0.25mM to 10 mM.

18. The method of claim 15, wherein the concentration of epigallocatechin gallate or a derivative thereof in the first reagent is between 0.5mM and 5 mM.

19. The method of any one of claims 14 to 18, wherein the formulated working liquid is allowed to stand for 2 days or more before being added to the sample to be stained.

20. The method of claim 19, wherein the formulated working fluid is allowed to stand for 3 days or more before being added to the sample to be stained.

21. The method of claim 19, wherein the formulated working fluid is allowed to sit for 7 days or more before being added to the sample to be stained.