Hematoxylin staining solution as well as preparation method and staining method thereof

Abstract

The invention relates to the technical field of biology, and particularly provides a hematoxylin staining solution and a preparation and staining method thereof.The hematoxylin staining solution is prepared from hematoxylin, absolute ethyl alcohol, aluminum potassium sulfate, sodium iodate, glacial acetic acid and water, the proportion of all the raw materials is adjusted in a targeted mode, the dosage of all the raw materials is strictly controlled, and it is accidentally found that the hematoxylin staining solution can be used for staining hematoxylin. After a tissue slice is stained by the hematoxylin staining solution provided by the invention, cell nucleus coloring is good, a tissue structure is clear, the tissue slice subjected to eosin restaining is bright in color, strong in contrast, easy to observe, ideal in staining effect, high in staining stability, not easy to fade and beneficial to long-term preservation of the slice, the preparation method is simple, and an unexpected effect is achieved.

Description

Technical Field

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

[0002] Hematoxylin is a natural, basic dye with a molecular formula of C 16 H 14 O6, molecular weight 302.288. It is mainly extracted from the core of a tree called "hematoxylon campechianum" native to Central and South America. Hematoxylin itself has no chromophore and does not have staining properties. It needs to be naturally or artificially oxidized and matured into hematoxylin red before it has staining effectiveness. Hematoxylin staining solution is the most commonly used staining solution in histochemistry and immunohistochemistry. It is used in combination with eosin staining solution, referred to as HE staining method, which is the most widely used technical method in histology, embryology, and pathology. Hematoxylin staining solution is a key reagent in the HE staining process, and its quality determines the quality of a stained slide.

[0003] At present, the most commonly used hematoxylin staining solutions are Harris hematoxylin staining solution, Mayer hematoxylin staining solution and Gill hematoxylin staining solution. The formula and staining process are the same, but the advantages and disadvantages are also different. For example, Harris hematoxylin staining solution has the advantages of being ready for use, short staining time, and bright colors, but the disadvantages are that the use cycle is short and the replacement frequency is high when the number of stained slides is large; there is a metal-like peroxide film floating on the surface of the liquid, or there is a dark brown oxide, which contaminates the slides; the mercuric oxide in the formula is almost insoluble in water and is highly toxic, polluting the environment and endangering the health of the staff. The advantage of Mayer hematoxylin staining solution is that it does not need to be boiled during preparation, does not contain mercuric oxide, and does not require differentiation; but its slide staining time is slightly longer than Harris hematoxylin staining solution. The advantage of Gill hematoxylin staining solution is that it uses sodium iodate instead of mercuric oxide, which reduces pollution and requires differentiation, but the disadvantages are that the coloring of the cell nucleus is not bright enough, the reagent usage time is short, and the coloring ability is not balanced enough. Summary of the invention

[0004] The purpose of the present invention is to provide a hematoxylin staining solution and a preparation and use method thereof in view of the problems that the hematoxylin staining solution in the prior art has poor staining effect, long staining time and complicated staining operation steps. With hematoxylin, anhydrous ethanol, potassium aluminum sulfate, sodium iodate, glacial acetic acid and water as raw materials, by adjusting the ratio of each raw material in a targeted manner and strictly controlling the amount of each raw material, it is unexpectedly found that the hematoxylin staining solution provided by the present invention has good cell nucleus coloring and clear tissue structure, and the tissue slices after eosin counterstaining have bright colors and strong contrast, are easy to observe, have ideal staining effect, are not easy to fade, and are conducive to long-term storage of slices.

[0005] In order to achieve the above object, the technical solution adopted by the present invention is:

[0006] A hematoxylin staining solution, comprising the following raw materials in parts by weight:

[0007] 10-14 parts of hematoxylin, 550-600 parts of anhydrous ethanol, 90-110 parts of potassium aluminum sulfate, 1-1.4 parts of sodium iodate, 45-50 parts of glacial acetic acid; 1500-1800 parts of water.

[0008] The present invention provides a hematoxylin staining solution, which uses hematoxylin, anhydrous ethanol, potassium aluminum sulfate, sodium iodate, glacial acetic acid and water as raw materials. By targetedly adjusting the ratio of each raw material and strictly controlling the amount of each raw material, it is unexpectedly found that the hematoxylin staining solution provided by the present invention has good cell nucleus coloring and clear tissue structure, and the tissue sections after counter-staining with eosin have bright colors and strong contrast, which are easy to observe, and the staining effect is ideal, the staining stability is high, and it is not easy to fade, which is conducive to the long-term preservation of the sections, and an unexpected effect is achieved.

[0009] Furthermore, the hematoxylin staining solution is prepared by the following raw materials in parts by weight: 12-14 parts of hematoxylin, 580-600 parts of anhydrous ethanol, 100-110 parts of potassium aluminum sulfate, 1-1.2 parts of sodium iodate, 45-48 parts of glacial acetic acid, and 1600-1800 parts of water. Strictly controlling the dosage ratio of each raw material can make the prepared hematoxylin staining solution have a better staining effect.

[0010] Another object of the present invention is to provide a method for preparing the above-mentioned hematoxylin staining solution.

[0011] The preparation method of the above-mentioned hematoxylin staining solution comprises the following steps:

[0012] Step 1, dissolve hematoxylin in anhydrous ethanol, stir and dissolve to form liquid A; dissolve potassium aluminum sulfate in a portion of water, stir and dissolve to form liquid B; dissolve sodium iodate in the remaining water, stir and dissolve to form liquid C;

[0013] Step 2: Add liquid A to liquid B in small amounts several times under stirring and cool;

[0014] Step 3, add liquid C to the mixed solution obtained in step 2, stir; seal and let stand for a while;

[0015] Step 4: Filter the material obtained in step 3, then add glacial acetic acid to the filtrate, let it stand again, and obtain a hematoxylin staining solution.

[0016] The invention provides a method for preparing a hematoxylin staining solution, and the preparation process is simple and easy to control.

[0017] Furthermore, in step 3, the standing time is 36-48 hours.

[0018] Furthermore, in step 4, the time for standing still again is 5-7 days.

[0019] Another object of the present invention is to provide a staining method for the above-mentioned hematoxylin staining solution.

[0020] The staining method of the above-mentioned hematoxylin staining solution comprises the following steps:

[0021] S1. Dewax the paraffin sections to water; S2. Stain with hematoxylin staining solution and drain; S3. Place the sections in anhydrous ethanol for 25-35 seconds; S4. Place the sections in eosin solution for 25-35 seconds; S5. Place the sections in water for 25-35 seconds; S6. Place the sections in 95% ethanol I for 10-20 seconds; S7. Place the sections in 95% ethanol II for 10-20 seconds; S8. Place the sections in anhydrous ethanol I for 10-30 seconds; S9. Place the sections in anhydrous ethanol II for 10-30 seconds; S10. Place the sections in xylene I for 20-30 seconds; S11. Place the sections in xylene II for 30-60 seconds; S12. Take out the sections, dry them in an oven, and seal them with gum.

[0022] The staining method of the hematoxylin staining solution provided in the present application simplifies the operation steps compared with the traditional HE staining method, the staining time is relatively short, the amount of hematoxylin staining slices per unit volume is significantly increased, the increase rate can reach more than 50%, and the storage time is longer.

[0023] Furthermore, the specific operation of S1 is: placing the slices in: xylene a for 1-3 min, xylene b for 1-3 min, xylene c for 1-3 min, anhydrous ethanol a for 1-2 min, anhydrous ethanol b for 1-2 min, anhydrous ethanol c for 1-2 min; washing with water for 1-2 min.

[0024] Furthermore, the specific operation of S2 is: put the slice into the hematoxylin stain solution for staining for 6-10 minutes, take out the staining rack and drain; wash with water for 1-2 minutes, take out the staining rack and drain again

[0025] In summary, due to the adoption of the above technical solution, the beneficial effects of the present invention are:

[0026] 1. The present invention provides a hematoxylin staining solution, which uses hematoxylin, anhydrous ethanol, potassium aluminum sulfate, sodium iodate, glacial acetic acid and water as raw materials. By targetedly adjusting the ratio of each raw material and strictly controlling the amount of each raw material, it is unexpectedly found that after staining tissue sections with the hematoxylin staining solution provided by the present invention, the cell nucleus is well colored and the tissue structure is clear. The tissue sections after counter-staining with eosin are bright in color with strong contrast, easy to observe, and have ideal staining effect. The staining stability is high and it is not easy to fade, which is beneficial to the long-term preservation of the sections, and an unexpected effect has been achieved.

[0027] 2. The present invention provides a method for preparing a hematoxylin staining solution, and the preparation process is simple and easy to control.

[0028] 3. Compared with the traditional HE staining method, the staining method of the hematoxylin staining solution provided by the present invention simplifies the operation steps, has a relatively short staining time, significantly increases the amount of hematoxylin staining per unit volume, with an increase rate of more than 50%, and has a longer storage time. DETAILED DESCRIPTION

[0029] The present invention is described in detail below.

[0030] In order to make the purpose, technical solution and advantages of the present invention more clearly understood, the present invention is further described in detail below in conjunction with the embodiments. It should be understood that the specific embodiments described herein are only used to explain the present invention and are not used to limit the present invention.

[0031] Example 1

[0032] Hematoxylin staining solution ingredients:

[0033] Hematoxylin (SIMGA) 12.5g, anhydrous ethanol (Wuhan Zhongtian) 592.5g; potassium aluminum sulfate (Sinopharm) 100g, sodium iodate 1.1g, glacial acetic acid (Wuhan Zhongtian) 47.2g, distilled water 1650g.

[0034] Preparation of hematoxylin staining solution:

[0035] 1. Dissolve hematoxylin in anhydrous ethanol and stir until it is completely dissolved to form liquid A;

[0036] 2. Dissolve potassium aluminum sulfate in preheated distilled water (60°C) and stir until completely dissolved to form liquid B;

[0037] 3. Dissolve sodium iodate in preheated distilled water (100 ml) and stir until completely dissolved to form liquid C;

[0038] 4. Add liquid A to liquid B in batches, stirring while adding;

[0039] 5. When the mixture is cooled to 37°C, add solution C and stir for 5 minutes;

[0040] 6. Seal and leave overnight;

[0041] 7. Filter after 48 hours;

[0042] 8. Add glacial acetic acid;

[0043] 9. Leave it for a week;

[0044] 10. Repack and use.

[0045] The HE staining method and process of hematoxylin staining solution are as follows:

[0046] Experimental Materials

[0047] Xylene (Wuhan Zhongtian), anhydrous ethanol (Wuhan Zhongtian), hematoxylin staining solution, water-soluble eosin (BaSO), 95% ethanol (Wuhan Zhongtian).

[0048] instrument:

[0049] Fully automatic staining machine Leica ST 5010, electric heating blast drying oven 101A-1ET.

[0050] Staining steps:

[0051] Test tissues: tonsils, appendix, and cervical tissue.

[0052] S1. Dewax the paraffin sections to water; sequentially place the sections in: xylene a for 1-3 min, xylene b for 1-3 min, xylene c for 1-3 min, anhydrous ethanol a for 1-2 min, anhydrous ethanol b for 1-2 min, anhydrous ethanol c for 1-2 min; wash with water for 1-2 min.

[0053] S2. Use hematoxylin staining solution to stain and drain; put the slices into the hematoxylin staining solution for staining for 6-10 minutes, take out the staining rack and drain; wash with water for 1-2 minutes, take out the staining rack and drain again;

[0054] S3, place the slices in anhydrous ethanol for 25-35 seconds;

[0055] S4, place the slice in eosin solution for 25-35 seconds;

[0056] S5. Place the slices in water for 25-35 seconds;

[0057] S6. Place the slices in 95% ethanol I for 10-20 seconds;

[0058] S7, place the slices in 95% ethanol II for 10-20 seconds;

[0059] S8, place the slices in anhydrous ethanol I for 10-30 seconds;

[0060] S9, place the slices in anhydrous ethanol II for 10-30 seconds;

[0061] S10, placing the slice in xylene I for 20-30 seconds;

[0062] S11, place the slice in xylene II for 30-60 seconds;

[0063] S12. Take out the slices, place them in an oven to dry, and seal them with gum.

[0064] Example 1 The specific dyeing time is shown in Table 1.

[0065] Table 1 HE staining procedure of fully automatic staining machine

[0066] Step Stn stain name Time Exact 1 OVEN 64℃ 2min No 2 1 Xylene a 1min No 3 2 Xylene b 1min No 4 3 Xylene c 3min No 5 4 Anhydrous ethanol 1min No 6 5 Anhydrous ethanol b 1min No 7 6 Anhydrous ethanol 2min No 8 WASH1 Washing 2min No 9 7 Hematoxylin staining solution 8min Yes 10 WASH2 Washing 1min No 11 8 Anhydrous ethanol 30s No 12 9 Eosin solution 25s yes 13 WASH3 Washing 30s yes 14 10 95% Ethanol I 10s yes 15 11 95% Ethanol II 10s yes 16 12 Anhydrous ethanol 1 10s yes 17 13 Anhydrous ethanol II 30s yes 18 14 Xylene 1 30s yes 19 15 II 1min yes

[0067] Interpretation criteria

[0068] Eosin has three colors:

[0069] Dark red: muscle cells (smooth, skeletal, cardiac) and epithelial cytoplasm;

[0070] Light pink: collagen;

[0071] Bright orange: red blood cells (RBC).

[0072] Hematoxylin shows multi-color effect:

[0073] Red-purple: The nucleolus has a higher color (protein and RNA).

[0074] Strong blue: cell nucleus, calcium salt granules.

[0075] Weak blue: basophilic cytoplasmic granules, alkaline mucus, glandular cell cytoplasm, neuronal cell cytoplasm, cartilage matrix.

[0076] The optical microscopic structure of tissues and cells is clearly layered, and the cell nucleus and patina are still clearly stained after a large number of staining effects, which can well analyze the cell results; the cell nucleus is well stained and the tissue structure is clear. The tissue sections after eosin counterstaining are bright in color and have a strong contrast. Every 500ml of hematoxylin staining solution can dye 1800 to 2000 sections, which can increase the amount of sections by more than 50% compared with the conventional hematoxylin staining solution in the prior art. The storage time can reach 36 months, and the staining stability is good.

[0077] Example 2

[0078] Hematoxylin staining solution ingredients:

[0079] Hematoxylin (SIMGA) 10g, anhydrous ethanol (Wuhan Zhongtian) 600g; potassium aluminum sulfate (Sinopharm) 90g, sodium iodate 1.4g, glacial acetic acid (Wuhan Zhongtian) 45g, distilled water 1800g.

[0080] Preparation of hematoxylin staining solution:

[0081] 1. Dissolve hematoxylin in anhydrous ethanol and stir until it is completely dissolved to form liquid A;

[0082] 2. Dissolve potassium aluminum sulfate in preheated distilled water (60°C) and stir until completely dissolved to form liquid B;

[0083] 3. Dissolve sodium iodate in preheated distilled water (100 ml) and stir until completely dissolved to form liquid C;

[0084] 4. Add liquid A to liquid B in batches, stirring while adding;

[0085] 5. When the mixture is cooled to 37°C, add solution C and stir for 5 minutes;

[0086] 6. Seal and leave overnight;

[0087] 7. Filter after 36 hours;

[0088] 8. Add glacial acetic acid;

[0089] 9. Leave it for 5 days;

[0090] 10. Repack and use.

[0091] The HE staining method and process of hematoxylin staining solution are as follows:

[0092] Experimental Materials

[0093] Xylene (Wuhan Zhongtian), anhydrous ethanol (Wuhan Zhongtian), hematoxylin staining solution, water-soluble eosin (BaSO), 95% ethanol (Wuhan Zhongtian).

[0094] instrument:

[0095] Fully automatic staining machine Leica ST 5010, electric heating blast drying oven 101A-1ET.

[0096] Staining steps:

[0097] Test tissues: tonsils, appendix, and cervical tissue.

[0098] S1. Dewax the paraffin sections to water; sequentially place the sections in: xylene a for 1-3 min, xylene b for 1-3 min, xylene c for 1-3 min, anhydrous ethanol a for 1-2 min, anhydrous ethanol b for 1-2 min, anhydrous ethanol c for 1-2 min; wash with water for 1-2 min.

[0099] S2. Use hematoxylin staining solution to stain and drain; put the slices into the hematoxylin staining solution for staining for 6-10 minutes, take out the staining rack and drain; wash with water for 1-2 minutes, take out the staining rack and drain again;

[0100] S3, place the slices in anhydrous ethanol for 25-35 seconds;

[0101] S4, place the slice in eosin solution for 25-35 seconds;

[0102] S5. Place the slices in water for 25-35 seconds;

[0103] S6. Place the slices in 95% ethanol I for 10-20 seconds;

[0104] S7, place the slices in 95% ethanol II for 10-20 seconds;

[0105] S8, place the slices in anhydrous ethanol I for 10-30 seconds;

[0106] S9, place the slices in anhydrous ethanol II for 10-30 seconds;

[0107] S10, placing the slice in xylene I for 20-30 seconds;

[0108] S11, place the slice in xylene II for 30-60 seconds;

[0109] S12. Take out the slices, place them in an oven to dry, and seal them with gum.

[0110] Example 2 The specific dyeing time is shown in Table 2.

[0111] Table 2 HE staining procedure of fully automatic staining machine

[0112] Step Stn stain name Time Exact 1 OVEN 64℃ 2min No 2 1 Xylene a 2min No 3 2 Xylene b 2min No 4 3 Xylene c 2min No 5 4 Anhydrous ethanol 1min No 6 5 Anhydrous ethanol b 2min No 7 6 Anhydrous ethanol 1min No 8 WASH1 Washing 1min No 9 7 Hematoxylin staining solution 6min Yes 10 WASH2 Washing 2min No 11 8 Anhydrous ethanol 25s No 12 9 Eosin solution 35s yes 13 WASH3 Washing 35s yes 14 10 95% Ethanol I 10s yes 15 11 95% Ethanol II 20s yes 16 12 Anhydrous ethanol 1 10s yes 17 13 Anhydrous ethanol II 30s yes 18 14 Xylene 1 20s yes 19 15 II 30s yes

[0113] Interpretation criteria

[0114] Eosin has three colors:

[0115] Dark red: muscle cells (smooth, skeletal, cardiac) and epithelial cytoplasm;

[0116] Light pink: collagen;

[0117] Bright orange: red blood cells (RBC).

[0118] Hematoxylin shows multi-color effect:

[0119] Red-purple: The nucleolus has a higher color (protein and RNA).

[0120] Strong blue: cell nucleus, calcium salt granules.

[0121] Weak blue: basophilic cytoplasmic granules, alkaline mucus, glandular cell cytoplasm, neuronal cell cytoplasm, cartilage matrix.

[0122] The optical microscopic structure of tissues and cells is clearly layered, and the cell nucleus and patina are still clearly stained after a large number of staining effects, which can well analyze the cell results; the cell nucleus is well stained and the tissue structure is clear. The tissue sections after eosin counterstaining are bright in color and have a strong contrast. Every 500ml of hematoxylin staining solution can dye 1800 to 2000 sections, which can increase the amount of sections by more than 50% compared with the conventional hematoxylin staining solution in the prior art. The storage time can reach 36 months, and the staining stability is good.

[0123] Example 3

[0124] Hematoxylin staining solution ingredients:

[0125] Hematoxylin (SIMGA) 14g, anhydrous ethanol (Wuhan Zhongtian) 550g; potassium aluminum sulfate (Sinopharm) 110g, sodium iodate 1g, glacial acetic acid (Wuhan Zhongtian) 50g, distilled water 1500g.

[0126] Preparation of hematoxylin staining solution:

[0127] 1. Dissolve hematoxylin in anhydrous ethanol and stir until it is completely dissolved to form liquid A;

[0128] 2. Dissolve potassium aluminum sulfate in preheated distilled water (60°C) and stir until completely dissolved to form liquid B;

[0129] 3. Dissolve sodium iodate in preheated distilled water (100 ml) and stir until completely dissolved to form liquid C;

[0130] 4. Add liquid A to liquid B in batches, stirring while adding;

[0131] 5. When the mixture is cooled to 37°C, add solution C and stir for 5 minutes;

[0132] 6. Seal and leave overnight;

[0133] 7. Filter after 48 hours;

[0134] 8. Add glacial acetic acid;

[0135] 9. Leave it for a week;

[0136] 10. Repack and use.

[0137] The HE staining method and process of hematoxylin staining solution are as follows:

[0138] Experimental Materials

[0139] Xylene (Wuhan Zhongtian), anhydrous ethanol (Wuhan Zhongtian), hematoxylin staining solution, water-soluble eosin (BaSO), 95% ethanol (Wuhan Zhongtian).

[0140] instrument:

[0141] Fully automatic staining machine Leica ST 5010, electric heating blast drying oven 101A-1ET.

[0142] Staining steps:

[0143] Test tissues: tonsils, appendix, and cervical tissue.

[0144] S1. Dewax the paraffin sections to water; sequentially place the sections in: xylene a for 1-3 min, xylene b for 1-3 min, xylene c for 1-3 min, anhydrous ethanol a for 1-2 min, anhydrous ethanol b for 1-2 min, anhydrous ethanol c for 1-2 min; wash with water for 1-2 min.

[0145] S2. Use hematoxylin staining solution to stain and drain; put the slices into the hematoxylin staining solution for staining for 6-10 minutes, take out the staining rack and drain; wash with water for 1-2 minutes, take out the staining rack and drain again;

[0146] S3, place the slices in anhydrous ethanol for 25-35 seconds;

[0147] S4, place the slice in eosin solution for 25-35 seconds;

[0148] S5. Place the slices in water for 25-35 seconds;

[0149] S6. Place the slices in 95% ethanol I for 10-20 seconds;

[0150] S7, place the slices in 95% ethanol II for 10-20 seconds;

[0151] S8, place the slices in anhydrous ethanol I for 10-30 seconds;

[0152] S9, place the slices in anhydrous ethanol II for 10-30 seconds;

[0153] S10, placing the slice in xylene I for 20-30 seconds;

[0154] S11, place the slice in xylene II for 30-60 seconds;

[0155] S12. Take out the slices, place them in an oven to dry, and seal them with gum.

[0156] The specific dyeing time of Example 3 is shown in Table 3.

[0157] Table 3 HE staining procedure of fully automatic staining machine

[0158]

[0159]

[0160] Interpretation criteria

[0161] Eosin has three colors:

[0162] Dark red: muscle cells (smooth, skeletal, cardiac) and epithelial cytoplasm;

[0163] Light pink: collagen;

[0164] Bright orange: red blood cells (RBC).

[0165] Hematoxylin shows multi-color effect:

[0166] Red-purple: The nucleolus has a higher color (protein and RNA).

[0167] Strong blue: cell nucleus, calcium salt granules.

[0168] Weak blue: basophilic cytoplasmic granules, alkaline mucus, glandular cell cytoplasm, neuronal cell cytoplasm, cartilage matrix.

[0169] The optical microscopic structure of tissues and cells is clearly layered, and the cell nucleus and patina are still clearly stained after a large number of staining effects, which can well analyze the cell results; the cell nucleus is well stained and the tissue structure is clear. The tissue sections after eosin counterstaining are bright in color and have a strong contrast. Every 500ml of hematoxylin staining solution can dye 1800 to 2000 sections, which can increase the amount of sections by more than 50% compared with the conventional hematoxylin staining solution in the prior art. The storage time can reach 36 months, and the staining stability is good.

[0170] Comparative Example 1

[0171] Compared with Example 1, the addition ratio of glacial acetic acid in the hematoxylin staining solution was adjusted in Comparative Example 1, and the rest of the preparation and staining process was consistent with Example 1.

[0172] Hematoxylin staining solution ingredients:

[0173] Hematoxylin (SIMGA) 12.5g, anhydrous ethanol (Wuhan Zhongtian) 592.5g; potassium aluminum sulfate (Sinopharm) 100g, sodium iodate 1.1g, glacial acetic acid (Wuhan Zhongtian) 30g, distilled water 1650g.

[0174] Preparation of hematoxylin staining solution:

[0175] 1. Dissolve hematoxylin in anhydrous ethanol and stir until it is completely dissolved to form liquid A;

[0176] 2. Dissolve potassium aluminum sulfate in preheated distilled water (60°C) and stir until completely dissolved to form liquid B;

[0177] 3. Dissolve sodium iodate in preheated distilled water (100 ml) and stir until completely dissolved to form liquid C;

[0178] 4. Add liquid A to liquid B in batches, stirring while adding;

[0179] 5. When the mixture is cooled to 37°C, add solution C and stir for 5 minutes;

[0180] 6. Seal and leave overnight;

[0181] 7. Filter after 48 hours;

[0182] 8. Add glacial acetic acid;

[0183] 9. Leave it for a week;

[0184] 10. Repack and use.

[0185] The HE staining method and process of hematoxylin staining solution are as follows:

[0186] Experimental Materials

[0187] Xylene (Wuhan Zhongtian), anhydrous ethanol (Wuhan Zhongtian), hematoxylin staining solution, water-soluble eosin (BaSO), 95% ethanol (Wuhan Zhongtian).

[0188] instrument:

[0189] Fully automatic staining machine Leica ST 5010, electric heating blast drying oven 101A-1ET.

[0190] Staining steps:

[0191] Test tissues: tonsils, appendix, and cervical tissue.

[0192] S1. Dewax the paraffin sections to water; sequentially place the sections in: xylene a for 1-3 min, xylene b for 1-3 min, xylene c for 1-3 min, anhydrous ethanol a for 1-2 min, anhydrous ethanol b for 1-2 min, anhydrous ethanol c for 1-2 min; wash with water for 1-2 min.

[0193] S2. Use hematoxylin staining solution to stain and drain; put the slices into the hematoxylin staining solution for staining for 6-10 minutes, take out the staining rack and drain; wash with water for 1-2 minutes, take out the staining rack and drain again;

[0194] S3, place the slices in anhydrous ethanol for 25-35 seconds;

[0195] S4, place the slice in eosin solution for 25-35 seconds;

[0196] S5. Place the slices in water for 25-35 seconds;

[0197] S6. Place the slices in 95% ethanol I for 10-20 seconds;

[0198] S7, place the slices in 95% ethanol II for 10-20 seconds;

[0199] S8, place the slices in anhydrous ethanol I for 10-30 seconds;

[0200] S9, place the slices in anhydrous ethanol II for 10-30 seconds;

[0201] S10, placing the slice in xylene I for 20-30 seconds;

[0202] S11, place the slice in xylene II for 30-60 seconds;

[0203] S12, taking out the slices, placing them in an oven to dry, and sealing them with gum. The specific staining time of Comparative Example 1 is shown in Table 4.

[0204] Table 4 HE dyeing procedure of fully automatic dyeing machine

[0205]

[0206]

[0207] The optical microscopic structure of tissues and cells is blurred, and the clarity of the staining of cell nuclei and patina in a large number of staining slides is not high, making it difficult to analyze cell results well.

[0208] Comparative Example 2

[0209] Compared with Example 1, Comparative Example 2 adjusted the addition ratio of glacial acetic acid in the hematoxylin staining solution, and the rest of the preparation and staining process were consistent with Example 1.

[0210] Hematoxylin staining solution ingredients:

[0211] Hematoxylin (SIMGA) 12.5g, anhydrous ethanol (Wuhan Zhongtian) 592.5g; potassium aluminum sulfate (Sinopharm) 100g, sodium iodate 1.1g, glacial acetic acid (Wuhan Zhongtian) 60g, distilled water 1650g.

[0212] Preparation of hematoxylin staining solution:

[0213] 1. Dissolve hematoxylin in anhydrous ethanol and stir until it is completely dissolved to form liquid A;

[0214] 2. Dissolve potassium aluminum sulfate in preheated distilled water (60°C) and stir until completely dissolved to form liquid B;

[0215] 3. Dissolve sodium iodate in preheated distilled water (100 ml) and stir until completely dissolved to form liquid C;

[0216] 4. Add liquid A to liquid B in batches, stirring while adding;

[0217] 5. When the mixture is cooled to 37°C, add solution C and stir for 5 minutes;

[0218] 6. Seal and leave overnight;

[0219] 7. Filter after 48 hours;

[0220] 8. Add glacial acetic acid;

[0221] 9. Leave it for a week;

[0222] 10. Repack and use.

[0223] The HE staining method and process of hematoxylin staining solution are as follows:

[0224] Experimental Materials

[0225] Xylene (Wuhan Zhongtian), anhydrous ethanol (Wuhan Zhongtian), hematoxylin staining solution, water-soluble eosin (BaSO), 95% ethanol (Wuhan Zhongtian).

[0226] instrument:

[0227] Fully automatic staining machine Leica ST 5010, electric heating blast drying oven 101A-1ET.

[0228] Staining steps:

[0229] Test tissues: tonsils, appendix, and cervical tissue.

[0230] S1. Dewax the paraffin sections to water; sequentially place the sections in: xylene a for 1-3 min, xylene b for 1-3 min, xylene c for 1-3 min, anhydrous ethanol a for 1-2 min, anhydrous ethanol b for 1-2 min, anhydrous ethanol c for 1-2 min; wash with water for 1-2 min.

[0231] S2. Use hematoxylin staining solution to stain and drain; put the slices into the hematoxylin staining solution for staining for 6-10 minutes, take out the staining rack and drain; wash with water for 1-2 minutes, take out the staining rack and drain again;

[0232] S3, place the slices in anhydrous ethanol for 25-35 seconds;

[0233] S4, place the slice in eosin solution for 25-35 seconds;

[0234] S5. Place the slices in water for 25-35 seconds;

[0235] S6. Place the slices in 95% ethanol I for 10-20 seconds;

[0236] S7, place the slices in 95% ethanol II for 10-20 seconds;

[0237] S8, place the slices in anhydrous ethanol I for 10-30 seconds;

[0238] S9, place the slices in anhydrous ethanol II for 10-30 seconds;

[0239] S10, placing the slice in xylene I for 20-30 seconds;

[0240] S11, place the slice in xylene II for 30-60 seconds;

[0241] S12. Take out the slices, place them in an oven to dry, and seal them with gum.

[0242] The specific dyeing time of Comparative Example 2 is shown in Table 5.

[0243] Table 5 HE dyeing procedure of fully automatic dyeing machine

[0244]

[0245]

[0246] The optical microscopic structure of tissues and cells is blurred, and the clarity of the staining of cell nuclei and patina in a large number of staining slides is not high, making it difficult to analyze cell results well.

[0247] Comparative Example 3

[0248] Compared with Example 1, Comparative Example 3 adjusted the addition ratio of each raw material in the hematoxylin staining solution, and the rest of the preparation and staining process were consistent with Example 1.

[0249] Hematoxylin staining solution ingredients:

[0250] Hematoxylin (SIMGA) 8g, anhydrous ethanol (Wuhan Zhongtian) 700g; potassium aluminum sulfate (Sinopharm) 50g, sodium iodate 1.8g, glacial acetic acid (Wuhan Zhongtian) 47.2g, distilled water 1650g.

[0251] Preparation of hematoxylin staining solution:

[0252] 1. Dissolve hematoxylin in anhydrous ethanol and stir until it is completely dissolved to form liquid A;

[0253] 2. Dissolve potassium aluminum sulfate in preheated distilled water (60°C) and stir until completely dissolved to form liquid B;

[0254] 3. Dissolve sodium iodate in preheated distilled water (100 ml) and stir until completely dissolved to form liquid C;

[0255] 4. Add liquid A to liquid B in batches, stirring while adding;

[0256] 5. When the mixture is cooled to 37°C, add solution C and stir for 5 minutes;

[0257] 6. Seal and leave overnight;

[0258] 7. Filter after 48 hours;

[0259] 8. Add glacial acetic acid;

[0260] 9. Leave it for a week;

[0261] 10. Repack and use.

[0262] The HE staining method and process of hematoxylin staining solution are as follows:

[0263] Experimental Materials

[0264] Xylene (Wuhan Zhongtian), anhydrous ethanol (Wuhan Zhongtian), hematoxylin staining solution, water-soluble eosin (BaSO), 95% ethanol (Wuhan Zhongtian).

[0265] instrument:

[0266] Fully automatic staining machine Leica ST 5010, electric heating blast drying oven 101A-1ET.

[0267] Staining steps:

[0268] Test tissues: tonsils, appendix, and cervical tissue.

[0269] S1. Dewax the paraffin sections to water; sequentially place the sections in: xylene a for 1-3 min, xylene b for 1-3 min, xylene c for 1-3 min, anhydrous ethanol a for 1-2 min, anhydrous ethanol b for 1-2 min, anhydrous ethanol c for 1-2 min; wash with water for 1-2 min.

[0270] S2. Use hematoxylin staining solution to stain and drain; put the slices into the hematoxylin staining solution for staining for 6-10 minutes, take out the staining rack and drain; wash with water for 1-2 minutes, take out the staining rack and drain again;

[0271] S3, place the slices in anhydrous ethanol for 25-35 seconds;

[0272] S4, place the slice in eosin solution for 25-35 seconds;

[0273] S5. Place the slices in water for 25-35 seconds;

[0274] S6. Place the slices in 95% ethanol I for 10-20 seconds;

[0275] S7, place the slices in 95% ethanol II for 10-20 seconds;

[0276] S8, place the slices in anhydrous ethanol I for 10-30 seconds;

[0277] S9, place the slices in anhydrous ethanol II for 10-30 seconds;

[0278] S10, placing the slice in xylene I for 20-30 seconds;

[0279] S11, place the slice in xylene II for 30-60 seconds;

[0280] S12. Take out the slices, place them in an oven to dry, and seal them with gum.

[0281] The specific dyeing time of Comparative Example 3 is shown in Table 6.

[0282] Table 6 HE staining procedure of fully automatic staining machine

[0283] Step Stn stain name Time 1 OVEN 64℃ 2min 2 1 Xylene a 1min 3 2 Xylene b 1min 4 3 Xylene c 3min 5 4 Anhydrous ethanol 1min 6 5 Anhydrous ethanol b 1min 7 6 Anhydrous ethanol 2min 8 WASH1 Washing 2min 9 7 Hematoxylin staining solution 8min 10 WASH2 Washing 1min 11 8 Anhydrous ethanol 30s 12 9 Eosin solution 25s 13 WASH3 Washing 30s 14 10 95% Ethanol I 10s 15 11 95% Ethanol II 10s 16 12 Anhydrous ethanol 1 10s 17 13 Anhydrous ethanol II 30s 18 14 Xylene 1 30s 19 15 II 1min

[0284] The optical microscopic structure of tissues and cells is blurred, and the clarity of the staining of cell nuclei and patina in a large number of staining slides is not high, making it difficult to analyze cell results well.

[0285] Comparative Example 4

[0286] Compared with Example 1, Comparative Example 4 adjusted the addition ratio of each raw material in the hematoxylin staining solution, and the rest of the preparation and staining process were consistent with Example 1.

[0287] Hematoxylin staining solution ingredients:

[0288] Hematoxylin (SIMGA) 18g, anhydrous ethanol (Wuhan Zhongtian) 400g; potassium aluminum sulfate (Sinopharm) 120g, sodium iodate 0.5g, glacial acetic acid (Wuhan Zhongtian) 47.2g, distilled water 1650g.

[0289] Preparation of hematoxylin staining solution:

[0290] 1. Dissolve hematoxylin in anhydrous ethanol and stir until it is completely dissolved to form liquid A;

[0291] 2. Dissolve potassium aluminum sulfate in preheated distilled water (60°C) and stir until completely dissolved to form liquid B;

[0292] 3. Dissolve sodium iodate in preheated distilled water (100 ml) and stir until completely dissolved to form liquid C;

[0293] 4. Add liquid A to liquid B in batches, stirring while adding;

[0294] 5. When the mixture is cooled to 37°C, add solution C and stir for 5 minutes;

[0295] 6. Seal and leave overnight;

[0296] 7. Filter after 48 hours;

[0297] 8. Add glacial acetic acid;

[0298] 9. Leave it for a week;

[0299] 10. Repack and use.

[0300] The HE staining method and process of hematoxylin staining solution are as follows:

[0301] Experimental Materials

[0302] Xylene (Wuhan Zhongtian), anhydrous ethanol (Wuhan Zhongtian), hematoxylin staining solution, water-soluble eosin (BaSO), 95% ethanol (Wuhan Zhongtian).

[0303] instrument:

[0304] Fully automatic staining machine Leica ST 5010, electric heating blast drying oven 101A-1ET.

[0305] Staining steps:

[0306] Test tissues: tonsils, appendix, and cervical tissue.

[0307] S1. Dewax the paraffin sections to water; sequentially place the sections in: xylene a for 1-3 min, xylene b for 1-3 min, xylene c for 1-3 min, anhydrous ethanol a for 1-2 min, anhydrous ethanol b for 1-2 min, anhydrous ethanol c for 1-2 min; wash with water for 1-2 min.

[0308] S2. Use hematoxylin staining solution to stain and drain; put the slices into the hematoxylin staining solution for staining for 6-10 minutes, take out the staining rack and drain; wash with water for 1-2 minutes, take out the staining rack and drain again;

[0309] S3, place the slices in anhydrous ethanol for 25-35 seconds;

[0310] S4, place the slice in eosin solution for 25-35 seconds;

[0311] S5. Place the slices in water for 25-35 seconds;

[0312] S6. Place the slices in 95% ethanol I for 10-20 seconds;

[0313] S7, place the slices in 95% ethanol II for 10-20 seconds;

[0314] S8, place the slices in anhydrous ethanol I for 10-30 seconds;

[0315] S9, place the slices in anhydrous ethanol II for 10-30 seconds;

[0316] S10, placing the slice in xylene I for 20-30 seconds;

[0317] S11, place the slice in xylene II for 30-60 seconds;

[0318] S12. Take out the slices, place them in an oven to dry, and seal them with gum.

[0319] The specific dyeing time of Comparative Example 4 is shown in Table 7.

[0320] Table 7 HE dyeing procedure of fully automatic dyeing machine

[0321]

[0322]

[0323] The optical microscopic structure of tissues and cells is blurred, and the clarity of the staining of cell nuclei and patina in a large number of staining slides is not high, making it difficult to analyze cell results well.

[0324] It can be seen that the hematoxylin staining solution provided by the present invention uses hematoxylin, anhydrous ethanol, potassium aluminum sulfate, sodium iodate, glacial acetic acid and water as raw materials. By targetedly adjusting the ratio of each raw material and strictly controlling the amount of each raw material, it is unexpectedly found that after the hematoxylin staining solution provided by the present invention is used to stain tissue sections, the cell nuclei are well colored and the tissue structure is clear. The tissue sections after counter-staining with eosin are bright in color and have a strong contrast, which is easy to observe. The staining effect is ideal, the staining stability is high, and it is not easy to fade, which is beneficial to the long-term preservation of the sections, and unexpected effects have been achieved.

[0325] The above description is only a preferred embodiment of the present invention and is not intended to limit the present invention. Any modifications, equivalent substitutions and improvements made within the spirit and principles of the present invention should be included in the protection scope of the present invention.

Claims

1. A hematoxylin staining solution, characterized in that The preparation comprises the following raw materials in parts by weight: 10-14 parts of hematoxylin, 550-600 parts of anhydrous ethanol, 90-110 parts of potassium aluminum sulfate, 1-1.4 parts of sodium iodate, 45-50 parts of glacial acetic acid; 1500-1800 parts of water.

2. The hematoxylin staining solution according to claim 1, characterized in that The invention is prepared from the following raw materials in parts by weight: 12-14 parts of hematoxylin, 580-600 parts of anhydrous ethanol, 100-110 parts of potassium aluminum sulfate, 1-1.2 parts of sodium iodate, 45-48 parts of glacial acetic acid, and 1600-1800 parts of water.

3. The method for preparing the hematoxylin staining solution according to claim 1 or 2, wherein: The following steps are involved: Step 1, dissolve hematoxylin in anhydrous ethanol, stir and dissolve to form liquid A; dissolve potassium aluminum sulfate in a portion of water, stir and dissolve to form liquid B; dissolve sodium iodate in the remaining water, stir and dissolve to form liquid C; Step 2: Add liquid A to liquid B in small amounts several times under stirring and cool; Step 3, add liquid C to the mixed solution obtained in step 2, stir; seal and let stand for a while; Step 4: Filter the material obtained in step 3, then add glacial acetic acid to the filtrate, let it stand again, and obtain a hematoxylin staining solution.

4. The method for preparing the hematoxylin staining solution according to claim 3, wherein In step 3, the standing time is 36-48 hours.

5. The method for preparing the hematoxylin staining solution according to claim 3, wherein In step 4, the time for standing still again is 5-7 days.

6. A method for dyeing using the hematoxylin staining solution as claimed in claim 1 or 2, characterized in that: The following steps are involved: S1. Dewax the paraffin sections to water; S2. Dye with hematoxylin staining solution and drain; S3. Place the slices in anhydrous ethanol for 25-35 seconds; S4, place the slices in eosin solution for 25-35 seconds; S5, place the slices in water for 25-35 seconds; S6, place the slices in 95% ethanol I for 10-20 seconds; S7, place the slices in 95% ethanol II for 10-20 seconds; S8, placing the slices in anhydrous ethanol I for 10-30 seconds; S9, placing the slices in anhydrous ethanol II for 10-30 seconds; S10, placing the slices in xylene I for 20-30 seconds; S11, placing the slices in xylene II for 30-60 seconds; S12, taking out the slices, placing them in an oven to dry, and sealing them with gum.

7. The method for dyeing with hematoxylin staining solution according to claim 6, characterized in that, The specific operation of S1 is: placing the slices in: xylene a for 1-3 min, xylene b for 1-3 min, xylene c for 1-3 min, anhydrous ethanol a for 1-2 min, anhydrous ethanol b for 1-2 min, anhydrous ethanol c for 1-2 min; washing with water for 1-2 min.

8. The method for dyeing with hematoxylin staining solution according to claim 6, characterized in that, The specific operation of S2 is: put the slices into the hematoxylin stain solution for staining for 6-10 minutes, take out the staining rack and drain; wash with water for 1-2 minutes, take out and stain again to drain.

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