A kind of hematoxylin staining solution and its preparation method and application

Abstract

The invention discloses a hematoxylin staining solution and a preparation method and application thereof. The hematoxylin staining solution comprises the following raw materials in mass percentage: 0.4%-0.6% hematoxylin, 0.02%-0.04% citric acid, 25%-30% diglycerol, 1.4%-1.8% acetic acid, 0.02%-0.04% aluminum oxide, 0.5%-0.7% sodium iodate, 16%-18% anhydrous ethanol, 0.8%-1.2% inert silicon dioxide, 2.8%-3.5% aluminum sulfate, and 44.12%-53.06% distilled water. The hematoxylin staining solution is simple to prepare and operate, durable, has good staining effect, and accurate results.

Description

Technical Field

[0001] The invention relates to the technical field of biomedicine, and in particular to a hematoxylin staining solution and a preparation method and application thereof. Background Art

[0002] Hematoxylin staining solution has been used in the field of biomedical technology for a long time. It is the most classic method for staining tissues and cells, and there is currently no better staining agent to replace it. The preparation of hematoxylin staining solution adopts two methods: natural oxidation and artificial oxidation.

[0003] Among them, although the natural oxidation method is relatively simple to operate, does not require a heating process, and can effectively avoid the problem of over-oxidation, natural oxidation takes a long time and cannot determine the oxidation maturity time, so it cannot meet the needs of clinical use. At present, the oxidant oxidation method mostly uses mercuric oxide, potassium permanganate, sodium iodate, and hydrogen peroxide, which can quickly oxidize hematoxylin to maturity and meet the timeliness of clinical use; however, there are many shortcomings. First, most of them are directly added with oxidants for oxidation. Due to the different purities of hematoxylin raw materials and oxidants, the amount of oxidants cannot be accurate, but after oxidation, the excess oxidants cannot be removed. During the storage of the product, other hydroxyls of hematoxylin are also oxidized to carbonyls and lose their staining ability; second, even if the oxidant is controlled below the consumption, the anti-peroxidation protective agent in most products cannot be closed from a molecular perspective. Therefore, the product will come into contact with oxygen during use or storage to produce a peroxide metal film, which is attached to the specimen and is difficult to clean, thus affecting the specimen staining quality; third, it is difficult to accurately control the temperature and time during the oxidation process, resulting in uneven product quality and affecting the staining effect. Summary of the invention

[0004] The purpose of the present invention is to provide a hematoxylin staining solution and a preparation method and application thereof, which can stain tissues and exfoliated cells quickly and effectively.

[0005] In one aspect of the present invention, the present invention provides a hematoxylin staining solution. According to an embodiment of the present invention, the hematoxylin staining solution includes the following components in mass percentage:

[0006]

[0007]

[0008] In addition, a hematoxylin staining solution according to the above embodiment of the present invention may also have the following additional technical features:

[0009] In some embodiments of the present invention, the hematoxylin staining solution is composed of the following raw materials in percentage by mass:

[0010]

[0011] In some embodiments of the present invention, the particle size of the inert silicon dioxide is 7000 mesh to 9000 mesh.

[0012] In another aspect of the present invention, the present invention provides a method for preparing a hematoxylin staining solution. According to an embodiment of the present invention, the preparation method comprises the following steps:

[0013] 1) Weigh hematoxylin and anhydrous ethanol into container 1, and stir to fully dissolve to obtain solution 1;

[0014] 2) Weigh diglycerol, aluminum sulfate, and half of distilled water into container 2, stir until fully dissolved, and then shake to obtain solution 2, which is a diglycerol solution that adsorbs metal aluminum ions;

[0015] 3) Weigh inert silicon dioxide, sodium iodate, and the other half of distilled water into container 3, stir until fully dissolved, and then shake to obtain solution 3;

[0016] 4) Weigh silicone oil and add it to the above solution 3, stir until fully dissolved, and then shake to obtain solution 4;

[0017] 5) Weigh citric acid and add it to the above solution 4, stir and fully dissolve to obtain solution 5, which is an inert oxidizing particle solution loaded with an oxidant;

[0018] 6) Pour the solution 1 into the solution 2, stirring while pouring to fully mix, then heat until boiling and stop heating, pour the solution 5 into the boiling solution while stirring with a glass rod, continue heating and boiling when no bubbles are generated, stop heating when the solution color turns dark purple, and cool to obtain solution 6;

[0019] 7) After filtering solution 6, weigh acetic acid, add it, and stir to mix thoroughly to obtain the hematoxylin staining solution.

[0020] In addition, the method for preparing a hematoxylin staining solution according to the above embodiment of the present invention may also have the following additional technical features:

[0021] In some embodiments of the present invention, stirring is performed using a glass rod.

[0022] In some embodiments of the present invention, shaking is performed using a constant temperature shaker, and in step 2), the shaking temperature is 35°C-40°C, and the shaking time is 1h-1.5h; in step 3), the shaking temperature is 53°C-60°C, and the shaking time is 1h-1.5h; in step 4), the shaking temperature is 53°C-60°C, and the shaking time is 25min-35min.

[0023] In some embodiments of the present invention, in step 6), heating is performed by an electric heating furnace, and cooling is performed by natural cooling or running water cooling.

[0024] In some embodiments of the present invention, in step 7), 0.8 micron filter paper is used for filtration.

[0025] In some embodiments of the present invention, in step 7), the hematoxylin staining solution is stored in a sealed bottle.

[0026] In another aspect of the present invention, the present invention provides a method for staining tissue or exfoliated cells. According to an embodiment of the present invention, the method comprises the following steps:

[0027] 1) Washing the dewaxed tissue sections or exfoliated cell smears;

[0028] 2) Then stain with hematoxylin for 3-5 minutes and rinse with tap water for 2-3 minutes;

[0029] 3) Differentiate with 1% hydrochloric acid ethanol solution for 3-5 seconds;

[0030] 4) Use lithium carbonate solution to blue for 30 seconds and wash with tap water for 1-2 minutes to complete the staining of tissues or exfoliated cells.

[0031] In addition, the method for staining tissue or exfoliated cells according to the above embodiment of the present invention may also have the following additional technical features:

[0032] In some embodiments of the present invention, in step 1), the dewaxing method of the slices is to dewax the paraffin slices in xylene I and II for 5-8 minutes each, and the washing method is to wash with anhydrous ethanol and 95% ethanol for 1-2 minutes each, then wash with 80% ethanol for 1-2 minutes, and rinse with tap water for 1-2 minutes;

[0033] In the step 3), the 1% hydrochloric acid ethanol solution is a mixed solution of 99 ml 75% ethanol and 1 ml concentrated hydrochloric acid;

[0034] In the step 4), the lithium carbonate solution is a mixed solution of 100 ml of distilled water and 13.3 g of lithium carbonate.

[0035] Compared with the prior art, the present invention has the following beneficial effects:

[0036] (1) The present invention prepares sodium iodate, silicone oil and inert silicon dioxide to form inert oxidized particles, and aluminum sulfate and diglycerol to form diglycerol for adsorbing metal aluminum ions. During the oxidation process, when a hydroxyl group on the hematoxylin molecule is oxidized to a carbonyl group, the carbonyl group quickly combines with the aluminum ion in the diglycerol for adsorbing metal aluminum ions. In this process, the four hydroxyl groups of diglycerol and the other four hydroxyl groups in the hematoxylin molecule are hydrogen-bonded to form a completely closed protective layer, which will no longer be oxidized by the oxidant. After the reaction is completed and cooled, the inert oxidized particles are filtered and removed. Therefore, it is ensured that the hematoxylin is completely oxidized and will not be over-oxidized during the preparation process, and the excess oxidant is removed to avoid damage to the dyeing solution during storage. The stability of the hematoxylin dyeing solution during preparation and long-term storage is protected from the two-way perspectives of the oxidant form and the formation of a diglycerol molecular protective layer. The preparation process of the present invention is not affected by difficult-to-control conditions such as oxidants, temperature, and time, which prolongs the storage stability time, improves the utilization rate, reduces the cost, and has a stable dyeing effect.

[0037] (2) During the staining process, the positively charged alkaline hematoxylin in the staining solution is firmly bonded to the negatively charged acidic DNA in the tissue or exfoliated cells through ionic bonds or hydrogen bonds, thereby being stained; in addition, citric acid and acetic acid are added to the staining solution to adjust the pH of the staining solution, effectively avoiding interference between the staining solution and the protein. After the tissue or exfoliated cells are stained, the cell nucleus appears blue-purple when observed under a microscope.

[0038] (3) The hematoxylin staining solution of the present invention can be stored for a long time. BRIEF DESCRIPTION OF THE DRAWINGS

[0039] Figure 1 This is a result diagram of staining cervical exfoliated cells on the day when the hematoxylin staining solution prepared in Example 2 of the present invention was prepared;

[0040] Figure 2 This is a result diagram of animal tissue sections stained with the hematoxylin staining solution prepared in Example 2 of the present invention on the day of preparation;

[0041] Figure 3 This is a result diagram of staining cervical exfoliated cells with the hematoxylin staining solution prepared in Example 2 of the present invention after 12 months;

[0042] Figure 4 This is a result diagram of animal tissue sections stained with the hematoxylin staining solution prepared in Example 2 of the present invention after 12 months;

[0043] Figure 5 This is a result diagram of staining cervical exfoliated cells with the hematoxylin staining solution prepared in Example 2 of the present invention after 24 months;

[0044] Figure 6This is a graph showing the result of staining animal tissue sections with the hematoxylin staining solution prepared in Example 2 of the present invention after 24 months.

[0045] Figure 7 This is a result diagram of staining cervical exfoliated cells on the day when the hematoxylin staining solution prepared in Example 3 of the present invention was prepared;

[0046] Figure 8 This is a result diagram of animal tissue sections stained with the hematoxylin staining solution prepared in Example 3 of the present invention on the day of preparation;

[0047] Fig. 9 This is a result diagram of staining cervical exfoliated cells with the hematoxylin staining solution prepared in Example 3 of the present invention after 12 months;

[0048] Fig.10 This is a result diagram of animal tissue sections stained with the hematoxylin staining solution prepared in Example 3 of the present invention after 12 months;

[0049] Fig.11 This is a result diagram of staining cervical exfoliated cells with the hematoxylin staining solution prepared in Example 3 of the present invention after 24 months;

[0050] Fig.12 This is a graph showing the result of staining animal tissue sections with the hematoxylin staining solution prepared in Example 3 of the present invention after 24 months.

[0051] Fig.13 This is a result diagram of staining cervical exfoliated cells on the day when the hematoxylin staining solution prepared in Example 4 of the present invention was prepared;

[0052] Fig.14 This is a result diagram of animal tissue sections stained with the hematoxylin staining solution prepared in Example 4 of the present invention on the day of preparation;

[0053] Fig.15 This is a result diagram of staining cervical exfoliated cells with the hematoxylin staining solution prepared in Example 4 of the present invention after 12 months;

[0054] Fig.16 This is a result diagram of animal tissue sections stained with the hematoxylin staining solution prepared in Example 4 of the present invention after 12 months;

[0055] Fig.17 This is a result diagram of staining cervical exfoliated cells with the hematoxylin staining solution prepared in Example 4 of the present invention after 24 months;

[0056] Fig.18 This is a graph showing the results of staining animal tissue sections with the hematoxylin staining solution prepared in Example 4 of the present invention after 24 months. DETAILED DESCRIPTION

[0057] The following will be combined with the drawings in the embodiments of the present invention to clearly and completely describe the technical solutions in the embodiments of the present invention. Obviously, the described embodiments are only part of the embodiments of the present invention, not all of the embodiments. Based on the embodiments of the present invention, all other embodiments obtained by ordinary technicians in this field without creative work are within the scope of protection of the present invention.

[0058] Example 1

[0059] A hematoxylin staining solution is composed of the following raw materials in percentage by mass (%):

[0060]

[0061]

[0062] Example 2

[0063] A method for preparing a hematoxylin staining solution comprises the following steps:

[0064] 1) Use an electronic balance to accurately weigh 0.5 g of hematoxylin and 17.5 g of anhydrous ethanol into a 500 ml conical flask. Stir gently with a glass rod until fully dissolved and cover with a stopper. This is solution 1.

[0065] 2) Use an electronic balance to accurately weigh 28 g of diglycerol, 3.1 g of aluminum sulfate, and 28.82 g of distilled water (half) into a 500 ml conical flask, stir with a glass rod to fully dissolve, place in a 37°C constant temperature shaker, shake slowly for 1 hour, and then take out to prepare a diglycerol solution adsorbing metal aluminum ions, which is solution 2;

[0066] 3) Use an electronic balance to accurately weigh 1.0 g of inert silica 8000 mesh, 0.6 g of sodium iodate, and 28.82 g of distilled water (half) into a 500 ml conical flask, stir with a glass rod to fully dissolve, place in a 56°C constant temperature shaker, shake slowly for 1 hour, and take out. This is solution 3;

[0067] 4) Accurately weigh 0.03 g of silicone oil using an electronic balance and add it to the above solution 3. Stir with a glass rod to fully dissolve it. Place it in a 56°C constant temperature shaker and shake it slowly for 30 minutes. Take it out. This is solution 4.

[0068] 5) Accurately weigh 0.03 g of citric acid using an electronic balance and add it to the above solution 4, stirring with a glass rod to fully dissolve, thereby preparing an oxidant-loaded inert oxidizing particle solution, which is solution 5;

[0069] 6) Slowly pour the solution 1 into the solution 2, stirring while pouring to fully mix, heat on an electric heating stove until boiling, remove from the electric heating stove, slowly pour the solution 5 while stirring with a glass rod, and continue to heat and boil in the electric heating stove when no bubbles are generated. When the color of the solution turns into dark purple-red, remove from the electric heating stove, cool naturally or cool in running water, and this is solution 6;

[0070] 7) After cooling, the solution 6 is filtered with 0.8 μm filter paper, 1.6 g of acetic acid is accurately weighed using an electronic balance and added, and stirred with a glass rod to mix thoroughly to obtain the hematoxylin staining solution, which is stored in a sealed bottle.

[0071] Example 3

[0072] A hematoxylin staining solution is composed of the following raw materials in percentage by mass (%):

[0073]

[0074]

[0075] The method for preparing a hematoxylin staining solution in Example 3 adopts the steps included in Example 2.

[0076] Example 4

[0077] A hematoxylin staining solution is composed of the following raw materials in percentage by mass (%):

[0078]

[0079] The method for preparing a hematoxylin staining solution in Example 4 adopts the steps included in Example 2.

[0080] Application Examples

[0081] The staining method for tissue and exfoliated cells comprises the following steps:

[0082] 1) Dewax the paraffin sections in xylene I and II for 7 minutes each to ensure full dewaxing;

[0083] 2) The dewaxed tissue sections or exfoliated cell smears were treated with absolute ethanol and 95% ethanol for 2 minutes each, then 80% ethanol for 1 minute, and then rinsed with tap water for 2 minutes;

[0084] 3) After 4 minutes of hematoxylin staining, rinse with tap water for 2 minutes;

[0085] 4) Differentiate with 1% hydrochloric acid ethanol solution for a few seconds (1% hydrochloric acid ethanol solution preparation: 99ml 75% ethanol plus 1ml concentrated hydrochloric acid);

[0086] 5) Use lithium carbonate solution (lithium carbonate solution preparation: add 13.3 grams of lithium carbonate into 100 ml of distilled water) for blueing for 30 seconds, and wash with tap water for 1 minute, thus completing the staining with the hematoxylin staining solution of the present invention.

[0087] During the staining process, the positively charged alkaline hematoxylin in the staining solution is firmly combined with the negatively charged acidic DNA in the tissue or exfoliated cells by ionic bonds or hydrogen bonds, thereby being stained; in addition, citric acid and acetic acid are added to the staining solution to adjust the pH of the staining solution, effectively avoiding interference between the staining solution and the protein. After staining, the cell nucleus of the tissue or exfoliated cells appears blue-purple under a microscope.

[0088] The hematoxylin staining solutions of Examples 2, 3, and 4 prepared on the same day, 12 months after preparation, and 24 months after preparation were taken to perform staining experiments on animal tissue sections and cervical exfoliated cell samples according to the above method.

[0089] like Figure 1 , Figure 2 , Figure 3 , Figure 4 , Figure 5 and Figure 6 As shown, there was no difference in the results of the hematoxylin staining solution obtained in Example 2 on the day of preparation, 12 months, and 24 months later.

[0090] like Figure 7 , Figure 8 , Fig. 9 , Fig.10 , Fig.11 and Fig.12 As shown, there was no difference in the results of the hematoxylin staining solution obtained in Example 3 on the day of preparation, 12 months, and 24 months later.

[0091] like Fig.13 , Fig.14 , Fig.15 , Fig.16 , Fig.17 and Fig.18 As shown, there was no difference in the results of the hematoxylin staining solution obtained in Example 4 on the day of preparation, 12 months, and 24 months later.

[0092] This indicates that the inert oxidizing particles of the present invention can oxidize hematoxylin and form a molecular protective layer with diglycerol to enclose the oxidized hematoxylin, so that the oxidized hematoxylin does not contact with external oxidizing substances (oxidants, dissolved oxygen), and the remaining hydroxyl groups of the oxidized hematoxylin are well protected, so that the effective ingredients of the hematoxylin dyeing solution prepared by the present invention are durable and stable.

[0093] The above contents are merely examples and explanations of the structure of the present invention. The technicians in this technical field may make various modifications or additions to the specific embodiments described or replace them in a similar manner. As long as they do not deviate from the structure of the present invention or exceed the scope defined by the claims, they should all fall within the protection scope of the present invention.

Claims

1. A hematoxylin staining solution, characterized in that The following components are included in mass percentage:

2. A hematoxylin staining solution according to claim 1, characterized in that, It is composed of the following raw materials in percentage by weight:

3. A hematoxylin staining solution according to claim 1 or 2, characterized in that: The particle size of the inert silicon dioxide is 7000 mesh to 9000 mesh.

4. A method for preparing the hematoxylin staining solution according to claim 1 or 2, characterized in that: The following steps are involved: 1) Weigh hematoxylin and anhydrous ethanol into container 1, and stir to fully dissolve to obtain solution 1; 2) Weigh diglycerol, aluminum sulfate, and half of distilled water into container 2, stir until fully dissolved, and then shake to obtain solution 2, which is a diglycerol solution that adsorbs metal aluminum ions; 3) Weigh inert silicon dioxide, sodium iodate, and the other half of distilled water into container 3, stir until fully dissolved, and then shake to obtain solution 3; 4) Weigh silicone oil and add it to the above solution 3, stir until fully dissolved, and then shake to obtain solution 4; 5) Weigh citric acid and add it to the above solution 4, stir and fully dissolve to obtain solution 5, which is an inert oxidizing particle solution loaded with an oxidant; 6) Pour the solution 1 into the solution 2, stirring while pouring to fully mix, then heat until boiling and stop heating, pour the solution 5 into the boiling solution while stirring with a glass rod, continue heating and boiling when no bubbles are generated, stop heating when the solution color turns dark purple, and cool to obtain solution 6; 7) After filtering solution 6, weigh acetic acid, add it, and stir to mix thoroughly to obtain the hematoxylin staining solution.

5. The method for preparing a hematoxylin staining solution according to claim 4, wherein: Stirring was performed using a glass rod.

6. The method for preparing a hematoxylin staining solution according to claim 4, wherein: The shaking is carried out on a constant temperature shaker. In the step 2), the shaking temperature is 35°C-40°C, and the shaking time is 1h-1.5h; in the step 3), the shaking temperature is 53°C-60°C, and the shaking time is 1h-1.5h; in the step 4), the shaking temperature is 53°C-60°C, and the shaking time is 25min-35min.

7. The method for preparing a hematoxylin staining solution according to claim 4, wherein: In the step 6), heating is performed by an electric heating furnace, and cooling is performed by natural cooling or running water cooling.

8. The method for preparing a hematoxylin staining solution according to claim 4, wherein: In the step 7), 0.8 micron filter paper is used for filtration, and the hematoxylin staining solution is stored in a sealed bottle.

9. A method for staining tissue or exfoliated cells, characterized in that: The following steps are involved: 1) Washing the dewaxed tissue sections or exfoliated cell smears; 2) then staining with the hematoxylin according to claim 1 or 2 for 3-5 minutes, and then rinsing with tap water for 2-3 minutes; 3) Differentiate with 1% hydrochloric acid ethanol solution for 3-5 seconds; 4) Use lithium carbonate solution to blue for 30 seconds and wash with tap water for 1-2 minutes to complete the staining of tissues or exfoliated cells.

10. A method for staining tissue or exfoliated cells according to claim 9, characterized in that: In the step 1), the dewaxing method of the slices is to dewax the paraffin slices in xylene I and II for 5-8 minutes each, and the washing method is to wash them with anhydrous ethanol and 95% ethanol for 1-2 minutes each, then wash them with 80% ethanol for 1-2 minutes, and rinse them with tap water for 1-2 minutes; In the step 3), the 1% hydrochloric acid ethanol solution is a mixed solution of 99 ml 75% ethanol and 1 ml concentrated hydrochloric acid; In the step 4), the lithium carbonate solution is a mixed solution of 100 ml of distilled water and 13.3 g of lithium carbonate.

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