The combined staining of Hematoxylin and Eosin, referred to as HE staining, is the most commonly used staining method in pathology and histology. Hematoxylin is a basic natural dye that can stain cell nuclei. The main component of chromatin in the nucleus is DNA. In the double-helix structure of DNA, the phosphate groups on the two nucleotide chains are outwardly oriented, making the outer side of the DNA double helix negatively charged and acidic. It easily binds to the positively charged basic hematoxylin dye through ionic bonds or hydrogen bonds and is thus stained.
Improved Harris Hematoxylin Staining Solution is an improvement on the classic Harris hematoxylin staining method. This staining solution is mercury-free, non-toxic, and free of oxide film. It stains nuclear chromatin deeply and delicately, and is often used clinically as a substitute for Harris hematoxylin staining solution. The staining time is generally 5-8 minutes. Its characteristics include a high degree of oxidation of hematoxylin and strong staining power. Although the staining time is short, it tends to overstain cell nuclei, cytoplasm, and fibers. After staining, hydrochloric acid-ethanol differentiation is required, making it a regressive staining method. This reagent is for research use only and not suitable for clinical diagnosis or other purposes.
Staining Principles
1. Nuclear Staining Principle: Hematoxylin is a basic natural dye capable of staining cell nuclei. The main component of chromatin in the nucleus is DNA. In the double-helix structure of DNA, the phosphate groups on the two nucleotide chains are outwardly directed, rendering the outer side of the DNA double helix negatively charged and acidic. It readily binds to the positively charged basic hematoxylin dye via ionic or hydrogen bonds, resulting in staining. Hematoxylin appears blue in alkaline solutions, thus staining the cell nucleus blue.
2. Cytoplasmic Staining Principle: Eosin is a chemically synthesized acidic dye that can stain cytoplasm under certain conditions. The main component of cytoplasm is protein, which is an amphoteric compound. The staining of cytoplasm is closely related to the pH value of the staining solution. When the pH value of the staining solution is below the isoelectric point (4.7-5.0) of cytoplasmic proteins, the cytoplasmic proteins ionize in a basic form, making the cytoplasm positively charged, which can then be stained by the negatively charged acidic dye. Eosin dissociates into negatively charged anions in water, which combine with the positively charged cations of cytoplasmic proteins to stain the cytoplasm red.
3. Differentiation: After staining, the process of removing excess bound stain from the tissue using specific solutions is called differentiation, and the solution used is called a differentiating solution. In HE staining, 0.5-1% hydrochloric acid-ethanol is commonly used as the differentiating solution. Acids can destroy the quinone structure of hematoxylin, causing the separation of tissue and pigment and subsequent decolorization. After hematoxylin staining, most tissues must undergo hydrochloric acid-ethanol differentiation to remove excess hematoxylin dye bound to the nucleus and hematoxylin dye adsorbed by the cytoplasm before eosin staining. This ensures clear differentiation between nuclear and cytoplasmic staining.
4. Blueing: After differentiation, hematoxylin exists in a red ionic state under acidic conditions (appearing red) and in a blue ionic state under alkaline conditions (appearing blue). Tissue sections stained red or pink after acidic ethanol differentiation must be immediately rinsed with water to stop differentiation, followed by treatment with weakly alkaline water to make the hematoxylin-stained nuclei appear blue. This process is called blueing. Alternatively, rinsing with tap water (especially warm water) can also achieve nuclear blueing, but it requires a longer time.
Materials to Be Prepared by the User
1. Hydrochloric acid-ethanol differentiating solution.
2. Blueing solution, such as dilute ammonia water, lithium carbonate solution, etc.
3. Graded ethanol.
4. Eosin staining solution.
5. 4% paraformaldehyde.
Operation Steps (For Reference Only)
(1) Paraffin Section Staining
1. Deparaffinization to Water
① Treat with xylene or Leagene Deparaffinization and Clearing Solution twice, 5-10 minutes each time.
② (Optional) Treat with absolute ethanol twice, 3-5 minutes each time.
③ 95% ethanol for 3-5 minutes.
④ 90% ethanol for 3-5 minutes.
⑤ 80% ethanol for 3-5 minutes.
⑥ Rinse with tap water or distilled water for 1-3 minutes.
2. Staining
① Stain with Improved Harris Hematoxylin Staining Solution for 3-8 minutes.
② Rinse with tap water or distilled water for 5-10 seconds.
③ (Optional) Differentiate with hydrochloric acid-ethanol for 2-5 seconds.
④ Rinse with tap water for 20-30 seconds.
⑤ (Optional) Blue with blueing solution for 20-40 seconds.
⑥ Rinse with tap water for 30-60 seconds.
⑦ Stain with eosin staining solution (water-soluble) for 0.5-2 minutes.
⑧ Rinse with tap water for 1-5 seconds.
3. Dehydration, Clearing, and Mounting
① 80% ethanol for 10-20 seconds.
② 90% ethanol for 10-20 seconds.
③ Treat with 95% ethanol twice, 1-2 minutes each time.
④ Treat with absolute ethanol twice, 2-3 minutes each time.
⑤ Clear with xylene three times, 2-3 minutes each time.
⑥ Mount with neutral resin.
Staining Results
Cell nuclei: blue.
Cytoplasm, muscle fibers, collagen fibers, etc.: red of varying intensities.
Keratin, red blood cells, etc.: bright orange-red.
(2) Frozen Section Staining
1. Fix with ether-ethanol mixed fixative for 5-10 seconds.
2. Rinse with tap water for 2-5 seconds.
3. Drop-stain with Improved Harris Hematoxylin Staining Solution for 1-2 minutes (may be heated to 50℃).
4. Rinse with tap water for 2-5 seconds.
5. (Optional) Differentiate with hydrochloric acid-ethanol for 2-5 seconds.
6. Rinse with tap water for 2-5 seconds.
7. Blue with blueing solution or warm water for 2-5 seconds.
8. Dehydration with 80% ethanol for 5-10 seconds
9. Stain with eosin staining solution for 2-5 seconds.
10. 80% ethanol for 1-2 seconds.
11. 95% ethanol for 1-2 seconds.
12. Absolute ethanol for 2-5 seconds.
13. Phenol-xylene (1:3) for 2-5 seconds.
14. Clear with xylene three times, 2-5 seconds each time.
15. Mount with neutral resin.
(3) Cell Staining
1. Fix with 4% paraformaldehyde for 10-20 minutes.
2. Rinse with tap water twice, 2 minutes each time.
3. Rinse with distilled water twice, 2 minutes each time.
4. Follow the staining, deparaffinization, clearing, and mounting steps for paraffin sections, but reduce the reaction time accordingly.
Staining Results
Cell nuclei: blue.
Cytoplasm, fibers: red.
Precautions
1. Ensure thorough deparaffinization of sections.
2. Regularly replace graded ethanol with fresh solutions.
3. The differentiation time with hydrochloric acid-ethanol depends on section thickness, tissue type, and reagent freshness. Additionally, rinse with tap water sufficiently after differentiation to completely remove acid.
4. The ether-ethanol mixed fixative is prepared by mixing equal volumes of ether and 95% ethanol, adding an appropriate amount of acetic acid, and storing in a sealed container.
5. Minimize staining time for frozen sections.
6. Common blueing solutions include 0.2-1% ammonia water, Scott's blueing solution, or 0.1-1% lithium carbonate solution.
7. For your safety and health, wear a lab coat and disposable gloves during operation.
8. This product is for scientific research use only. Any other uses are strictly prohibited.
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