A hematoxylin staining solution suitable for fully automatic immunohistochemical staining instrument and a preparation method thereof

Abstract

The invention discloses a hematoxylin staining solution suitable for a fully automatic immunohistochemical staining instrument and a preparation method thereof, wherein the hematoxylin staining solution comprises the following components by weight: 1 to 3 parts of hematoxylin, 10 to 14 parts of potassium aluminum sulfate, 80 to 120 parts of anhydrous ethanol, 600 to 800 parts of distilled water, 0.18 to 0.22 parts of sodium iodate, 0.4 to 0.6 parts of citric acid, and an appropriate amount of pH regulator to adjust the pH of the hematoxylin staining solution to 2.1 ± 0.2. The hematoxylin staining solution of the invention has a lower concentration than conventional hematoxylin staining solution, and a lower pH value than conventional hematoxylin staining solution. After the hematoxylin staining solution is used to redye the tissue, it only needs to be rinsed with a partial alkaline detergent to ensure that the tissue cell nucleus is colored light blue, which forms a sharp contrast with the DAB color, and the tissue layer is clear, thereby improving the contrast between the hematoxylin staining solution and the DAB color developer color, and helping the pathologist to interpret the fully automatic immunohistochemical results.

Description

Technical Field

[0001] The invention relates to the field of biotechnology, and in particular to a hematoxylin staining solution suitable for a full-automatic immunohistochemical staining instrument and a preparation method thereof. Background Art

[0002] In the fully automatic immunohistochemical staining technology, the coloring of the cell nucleus can directly affect the quality of the pathological section. The composition and quality of the hematoxylin staining solution determine the staining effect of the tissue cell nucleus, and the hematoxylin staining effect of the tissue cell nucleus determines the final hematoxylin staining effect and the quality of the section. At present, there are many types of hematoxylin staining solutions on the market, and the preparation methods are also diverse, but they all have the same defects, such as: ① The oxidant used in the preparation of hematoxylin staining solution is mercuric oxide, which is dangerous; ② It is easy to form an oxide film and hematoxylin precipitates when exposed to air; ③ The staining time is not fixed, ranging from a few seconds to a few minutes; therefore, when these hematoxylins are used in fully automatic immunohistochemical staining instruments, they often block the sample probe and the counterstaining color is lighter or darker. After manual hematoxylin counterstaining, 1% hydrochloric acid alcohol is often required for differentiation to remove the excessive hematoxylin stain bound to the cell nucleus and the hematoxylin stain adsorbed to the cytoplasm, so as to achieve a better staining effect; however, when the fully automatic immunohistochemical stainer uses hematoxylin stain for counterstaining, the differentiation step of the fully automatic immunohistochemical stainer is often unable to be achieved due to limitations such as the material of the sample pipe (alcohol can easily cause the plastic pipe to harden), the staining process, and the instrument running time, which easily leads to poor hematoxylin counterstaining effects.

[0003] In addition, most of the hematoxylin stains are only suitable for routine pathological staining such as hematoxylin-eosin staining. After re-staining, the cell nucleus is stained dark blue or dark purple (patent number: CN108362539A). In conventional pathological staining, its staining effect is generally recognized. However, in the field of fully automatic immunohistochemistry, after re-staining the tissue with the hematoxylin stain prepared by the above configuration method, the hematoxylin color of the tissue cells is dark blue-purple, and the contrast between it and the color of 3,3-diaminobenzidine tetrahydrochloride (DAB) presented by the antibody in the immunohistochemistry technology is low, resulting in a series of problems. For example, some weakly expressed antibodies are easily covered by the hematoxylin color due to the light color of DAB, which causes visual fatigue of the pathologist when interpreting the immunohistochemistry results, thereby affecting the pathologist's interpretation of the immunohistochemistry results. There are two reasons for the above problems: first, the amount of hematoxylin and potassium aluminum sulfate in the above hematoxylin preparation method is too much and the ratio is not good, resulting in a high concentration of the prepared hematoxylin dye solution; second, the pH value of the hematoxylin dye solution produced by the above preparation method is high, most of which is 2.8-3.5 (patent number: CN113218731A); these two reasons can lead to problems such as the cell nucleus being stained too dark blue-purple after staining with the hematoxylin dye solution, unclear tissue layers, and easy formation of an oxide film to block the sample addition pipeline. Summary of the invention

[0004] The technical problem to be solved by the present invention is to provide a hematoxylin dye solution suitable for a fully automatic immunohistochemical staining instrument and a preparation method thereof, so as to solve the problem that the hematoxylin dye solution prepared by the existing hematoxylin preparation method is not suitable for fully automatic immunohistochemical technology.

[0005] The technical problem to be solved by the present invention is achieved through the following technical solutions:

[0006] A hematoxylin staining solution suitable for a full-automatic immunohistochemical staining instrument comprises the following components in parts by weight: 1-3 parts of hematoxylin, 10-14 parts of potassium aluminum sulfate, 80-120 parts of anhydrous ethanol, 600-800 parts of distilled water, 0.18-0.22 parts of sodium iodate, 0.4-0.6 parts of citric acid, and an appropriate amount of pH regulator to adjust the pH of the hematoxylin staining solution to 2.1±0.2.

[0007] Preferably, the following components are included in parts by weight: 2 parts of hematoxylin, 12 parts of potassium aluminum sulfate, 100 parts of anhydrous ethanol, 700 parts of distilled water, 0.2 parts of sodium iodate, 0.5 parts of citric acid, and an appropriate amount of pH regulator to adjust the pH of the hematoxylin dye solution to 2.1.

[0008] A method for preparing a hematoxylin staining solution suitable for a fully automatic immunohistochemical staining instrument comprises the following steps:

[0009] (1) Weigh hematoxylin according to the measurement and completely dissolve it in anhydrous ethanol;

[0010] (2) Weigh potassium aluminum sulfate according to the measurement and dissolve it completely in distilled water;

[0011] (3) Pour the hematoxylin ethanol solution into the dissolved potassium aluminum sulfate solution and stir evenly;

[0012] (4) slowly add sodium iodate and stir to completely dissolve it;

[0013] (5) Add citric acid and stir to completely dissolve it;

[0014] (6) Use a pH adjuster to adjust the pH of the hematoxylin stain solution to 2.1 ± 0.2;

[0015] (7) Filter with filter paper or gauze to obtain the hematoxylin dye solution.

[0016] A method for dyeing using hematoxylin dye solution, comprising the following steps:

[0017] (1) The pre-treated tissue is paraffin-embedded and sliced, and then the slices are baked and stained using an automatic immunohistochemical staining instrument;

[0018] (2) Re-staining with hematoxylin solution for 3-8 min. After staining, the sections are rinsed with PBS. After the PBS rinse is completed, the operation of the fully automatic immunohistochemical staining instrument is terminated;

[0019] (3) Remove the slices from the fully automatic immunohistochemical staining instrument, wash them with tap water, wash them with anhydrous ethanol, dry them in an oven, and seal them with neutral gum.

[0020] Preferably, the staining time is 5 min.

[0021] Preferably, the slice thickness is 3 μm.

[0022] Preferably, the tissue type is tonsil tissue, and the primary antibodies selected for testing are CD68, CD3 and Pax-5.

[0023] The above technical solution of the present invention has the following beneficial effects:

[0024] (1) This formula is developed for fully automatic immunohistochemistry technology. The dosage of each chemical drug and reagent in the formula is reasonable, the preparation method is simple, no heating is required during the preparation process, and no waiting for oxidation after preparation is required. It is easy to use and has a long shelf life.

[0025] (2) The hematoxylin dye solution provided by the present invention has a lower concentration and a lower pH value than that prepared by the traditional formula, and is less likely to produce an oxide film and hematoxylin precipitation when exposed to air for a long time;

[0026] (3) The hematoxylin staining solution provided by the present invention uses citric acid instead of traditional glacial acetic acid, which has stronger selectivity for tissue cell nuclei, making the tissue structure clearer after re-staining with the hematoxylin staining solution;

[0027] (4) The hematoxylin provided by the present invention eliminates the glycerol commonly used in the traditional hematoxylin staining solution formula. Glycerol makes the hematoxylin staining solution viscous, deepens the coloring of the tissue sections to be tested, and requires enhanced differentiation after re-staining. The hematoxylin staining solution provided by the present invention does not have this defect, and tissue sections with good staining effect can be obtained without differentiation after re-staining;

[0028] (5) The hematoxylin stain provided by the present invention has good nucleus staining and clear tissue structure in tissue sections. The hematoxylin color is light blue and bright, with a high contrast with the DAB color in immunohistochemistry, making it easy to observe.

[0029] (6) The hematoxylin staining solution provided by the present invention has a re-staining time of 3-8 minutes, a relatively small time threshold, and is more suitable for the staining procedure of a fully automatic immunohistochemical staining instrument.

[0030] In summary, the present application changes the concentration of the hematoxylin stain by adjusting the dosage of hematoxylin and potassium aluminum sulfate, so that the nuclei are moderately stained and the tissue layers are clear after re-staining; moreover, the pH value of the prepared hematoxylin stain is adjusted with a pH regulator to appropriately reduce the binding ability of hematoxylin to tissue cells, so that the nuclei are brightly stained and the background color is clearer after re-staining, thereby improving the contrast between the hematoxylin color and the DAB color. BRIEF DESCRIPTION OF THE DRAWINGS

[0031] The accompanying drawings, which are incorporated in and constitute a part of the specification, illustrate embodiments of the invention and, together with the description, serve to explain the principles of the invention.

[0032] Figure 1 It is a comparison chart of the dyeing results of Examples 1-3 and Comparative Example 1. DETAILED DESCRIPTION

[0033] Various exemplary embodiments of the present invention will now be described in detail. It should be noted that the relative arrangement of components and steps, numerical expressions and numerical values ​​set forth in these embodiments do not limit the scope of the present invention unless otherwise specifically stated.

[0034] Example 1

[0035] Prepare hematoxylin stain:

[0036] (1) dissolving 3 g of hematoxylin in 80 mL of anhydrous ethanol to obtain a hematoxylin ethanol solution;

[0037] (2) dissolving 14 g of potassium aluminum sulfate in 600 mL of distilled water to obtain a potassium aluminum sulfate solution;

[0038] (3) mixing the hematoxylin ethanol solution obtained in (1) with the potassium aluminum sulfate solution obtained in (2) to obtain a mixed solution A;

[0039] (4) Add 220 mg of sodium iodate to the mixed solution A obtained in (3), and stir for 5 min to completely dissolve it, thereby obtaining a mixed solution B;

[0040] (5) Add 600 mg of citric acid to the mixed solution B obtained in (4), and stir to completely dissolve it to obtain a mixed solution C;

[0041] (6) The pH of the mixed solution C obtained in (5) was adjusted to 2.3 using a pH adjuster prepared in this laboratory;

[0042] (7) The pH-adjusted mixed solution is filtered through filter paper or gauze and can be used in a fully automatic immunohistochemical staining instrument;

[0043] Tissue staining: Tonsillar tissue was used, and the primary antibodies selected for testing were CD68, CD3, and Pax-5.

[0044] After a series of pretreatments, the tissue was paraffin-embedded and sliced ​​with a thickness of 3 μm. After the slices were baked according to the baking requirements of this laboratory, the staining test was performed using the laboratory's fully automatic immunohistochemical staining instrument.

[0045] The fully automatic hematoxylin counterstaining step uses the hematoxylin staining solution for counterstaining, the staining time is 5 minutes, and the sections are rinsed with PBS after staining. After the PBS rinse is completed, the operation of the fully automatic immunohistochemical staining instrument is completed, and the sections are removed from the fully automatic immunohistochemical staining instrument, and washed with tap water. The sections washed with tap water are washed with anhydrous ethanol, and the sections are placed in an oven to dry, and then sealed with neutral gum.

[0046] Example 2

[0047] Prepare hematoxylin stain:

[0048] (1) Dissolve 1 g of hematoxylin in 120 mL of anhydrous ethanol to obtain a hematoxylin ethanol solution;

[0049] (2) dissolving 10 g of potassium aluminum sulfate in 800 mL of distilled water to obtain a potassium aluminum sulfate solution;

[0050] (3) mixing the hematoxylin ethanol solution obtained in (1) with the potassium aluminum sulfate solution obtained in (2) to obtain a mixed solution A;

[0051] (4) Add 180 mg of sodium iodate to the mixed solution A obtained in (3), and stir for 5 min to completely dissolve it, thereby obtaining a mixed solution B;

[0052] (5) Add 400 mg of citric acid to the mixed solution B obtained in (4), and stir to completely dissolve it to obtain a mixed solution C;

[0053] (6) The pH of the mixed solution C obtained in (5) was adjusted to 1.9 using a pH adjuster prepared in this laboratory;

[0054] (7) filtering the pH-adjusted mixed solution with filter paper or gauze and then using it in an automatic immunohistochemical staining instrument;

[0055] Tissue staining: Tonsillar tissue was used, and the primary antibodies selected for testing were CD68, CD3, and Pax-5.

[0056] After a series of pretreatments, the tissue was paraffin-embedded and sliced ​​with a thickness of 3 μm. After the slices were baked according to the baking requirements of this laboratory, the staining test was performed using the laboratory's fully automatic immunohistochemical staining instrument.

[0057] The fully automatic hematoxylin counterstaining step uses the hematoxylin staining solution for counterstaining, the staining time is 5 minutes, and the sections are rinsed with PBS after staining. After the PBS rinse is completed, the operation of the fully automatic immunohistochemical staining instrument is completed, and the sections are removed from the fully automatic immunohistochemical staining instrument, and washed with tap water. The sections washed with tap water are washed with anhydrous ethanol, and the sections are placed in an oven to dry, and then sealed with neutral gum.

[0058] Example 3

[0059] Prepare hematoxylin stain:

[0060] (1) dissolving 2 g of hematoxylin in 100 mL of anhydrous ethanol to obtain a hematoxylin ethanol solution;

[0061] (2) adding 12 g of potassium aluminum sulfate to 700 mL of distilled water to obtain a potassium aluminum sulfate solution;

[0062] (3) mixing the hematoxylin ethanol solution obtained in (1) with the potassium aluminum sulfate solution obtained in (2) to obtain a mixed solution A;

[0063] (4) Add 200 mg of sodium iodate to the mixed solution A obtained in (3), stir for 5 min to completely dissolve it, and obtain a mixed solution B;

[0064] (5) Add 500 mg of citric acid to the mixed solution B obtained in (4), and stir to completely dissolve it to obtain a mixed solution C;

[0065] (6) The pH of the mixed solution C obtained in (5) was adjusted to 2.1 using a pH adjuster prepared in this laboratory;

[0066] (7) filtering the pH-adjusted mixed solution with filter paper or gauze and then using it in an automatic immunohistochemical staining instrument;

[0067] Tissue staining: Tonsillar tissue was used, and the primary antibodies selected for testing were CD68, CD3, and Pax-5.

[0068] After a series of pretreatments, the tissue was paraffin-embedded and sliced ​​with a thickness of 3 μm. After the slices were baked according to the baking requirements of this laboratory, the staining test was performed using the laboratory's fully automatic immunohistochemical staining instrument.

[0069] The fully automatic hematoxylin counterstaining step uses the hematoxylin staining solution for counterstaining, the staining time is 5 minutes, and the sections are rinsed with PBS after staining. After the PBS rinse is completed, the operation of the fully automatic immunohistochemical staining instrument is completed, and the sections are removed from the fully automatic immunohistochemical staining instrument, and washed with tap water. The sections washed with tap water are washed with anhydrous ethanol, and the sections are placed in an oven to dry, and then sealed with neutral gum.

[0070] Comparative Example 1

[0071] Prepare hematoxylin stain:

[0072] (1) dissolving 2 g of hematoxylin in 100 mL of anhydrous ethanol to obtain a hematoxylin ethanol solution;

[0073] (2) dissolving 12 g of potassium aluminum sulfate in 700 mL of distilled water to obtain a potassium aluminum sulfate solution;

[0074] (3) mixing the hematoxylin ethanol solution obtained in (1) with the potassium aluminum sulfate solution obtained in (2) to obtain a mixed solution A;

[0075] (4) Add 200 mg of sodium iodate to the mixed solution A obtained in (3), stir for 5 min to completely dissolve it, and obtain a mixed solution B;

[0076] (5) Add 500 mg of citric acid to the mixed solution B obtained in (4), and stir to completely dissolve it to obtain a mixed solution C;

[0077] (6) The pH value of the mixed solution was measured and the pH value was about 2.8;

[0078] (7) Filtering the mixed solution C directly with filter paper or gauze and then using it in an automatic immunohistochemical staining instrument;

[0079] Tissue staining: Tonsillar tissue was used, and the primary antibodies selected for testing were CD68, CD3, and Pax-5.

[0080] After a series of pretreatments, the tissue was paraffin-embedded and sliced ​​with a thickness of 3 μm. After the slices were baked according to the baking requirements of this laboratory, the staining test was performed using the laboratory's fully automatic immunohistochemical staining instrument.

[0081] The fully automatic hematoxylin counterstaining step uses the hematoxylin staining solution for counterstaining, the staining time is 5 minutes, and the sections are rinsed with PBS after staining. After the PBS rinse is completed, the operation of the fully automatic immunohistochemical staining instrument is completed, and the sections are removed from the fully automatic immunohistochemical staining instrument, and washed with tap water. The sections washed with tap water are washed with anhydrous ethanol, and the sections are placed in an oven to dry, and then sealed with neutral gum.

[0082] The dyeing effect of the tissues after Examples 1 to 3 and Comparative Example 1 is shown in the color chart. Figure 1 The dyeing effects of Examples 1 to 3 and Comparative Example 1 were compared and analyzed, and the results are shown in Table 1:

[0083] Table 1

[0084]

[0085]

[0086] The concentration of the hematoxylin dye solution prepared by the preparation method of the present invention is lower than that of the conventional hematoxylin dye solution, and the pH value is lower than that of the conventional hematoxylin dye solution, and the pH value is 2.1±0.2. After the tissue is redyeed with the hematoxylin dye solution, it only needs to be rinsed with a slightly alkaline detergent to ensure that the tissue cell nucleus is colored light blue, which forms a sharp contrast with the DAB color, and the tissue layer is clear, thereby improving the contrast between the hematoxylin dye solution and the DAB color developer color, and helping the pathologist to interpret the results of fully automatic immunohistochemistry.

[0087] Although the present invention has been disclosed as above by the embodiments, it is not intended to limit the present invention. Any person skilled in the art may make various choices and modifications without departing from the spirit and scope of the present invention. Therefore, the protection scope of the present invention is limited by the claims and their equivalents.

Claims

1. A method for staining using hematoxylin stain, wherein the hematoxylin stain is suitable for a fully automatic immunohistochemical staining instrument, characterized in that: The hematoxylin dye solution is composed of the following components in parts by weight: 2 parts of hematoxylin, 12 parts of potassium aluminum sulfate, 100 parts of anhydrous ethanol, 700 parts of distilled water, 0.2 parts of sodium iodate, 0.5 parts of citric acid, and an appropriate amount of pH regulator to adjust the pH of the hematoxylin dye solution to 2.1; The preparation method comprises the following steps: (1) Weigh hematoxylin according to the measurement and completely dissolve it in anhydrous ethanol; (2) Weigh potassium aluminum sulfate according to the measurement and dissolve it completely in distilled water; (3) Pour the hematoxylin ethanol solution into the dissolved potassium aluminum sulfate solution and stir evenly; (4) Slowly add sodium iodate and stir until it is completely dissolved; (5) Add citric acid and stir to completely dissolve; (6) Use a pH adjuster to adjust the pH of the hematoxylin stain solution to 2.1; (7) Filter with filter paper or gauze to obtain the hematoxylin dye solution; The method of staining with hematoxylin staining solution comprises the following steps: (1) The pre-treated tissue is embedded in paraffin and sliced, and then the slices are baked and stained using an automatic immunohistochemical staining instrument; (2) Use hematoxylin staining solution for counterstaining for 5 min. After staining, the sections are rinsed with PBS. After the PBS rinse is completed, the operation of the fully automatic immunohistochemical staining instrument is terminated; (3) Remove the slices from the fully automatic immunohistochemical staining instrument, wash them with tap water, wash them with anhydrous ethanol, dry them in an oven, and seal them with neutral gum.

2. The method for dyeing with hematoxylin solution according to claim 1, wherein In the method of staining with hematoxylin solution, the thickness of the slice in step (1) is 3 μm.

3. The method for dyeing with hematoxylin solution according to claim 1, wherein In the method of staining with hematoxylin solution, the tissue type in step (1) is tonsil tissue, and the primary antibodies selected for testing are CD68, CD3 and Pax-5.

4. Use of the hematoxylin stain according to claim 1 in a fully automatic immunohistochemical staining instrument.

Images