Hematoxylin-Eosin staining, commonly referred to as HE staining, is the most widely used staining method in pathology and histology. Hematoxylin is an alkaline natural dye that stains 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 face outward, making the outer side of the DNA double helix negatively charged and acidic. This allows it to easily bind to the positively charged alkaline hematoxylin dye via ionic or hydrogen bonds, resulting in staining.
Cole Hematoxylin Staining Solution is a chemically oxidized and mature alum hematoxylin, mainly composed of hematoxylin, potassium aluminum sulfate, glycerol, and other components. It contains a low concentration of hematoxylin and does not form an oxide film. It stains cell nuclei clearly without coloring the cytoplasm or fibrous components, and belongs to progressive staining—thus, hydrochloric acid-ethanol differentiation is not required after staining. The staining time is approximately 5-10 minutes, and it is mainly used for counterstaining cell nuclei.
Materials to Be Prepared by the User
1. Hydrochloric acid-ethanol differentiation solution
2. Bluing solution (e.g., dilute ammonia water, lithium carbonate solution, etc.)
3. Graded ethanol
4. Eosin staining solution
5. 4% paraformaldehyde
Operating Procedures (For Reference Only)
(1) Staining of Paraffin Sections
1. Deparaffinize sections to water
2. Staining
① Cole Hematoxylin Staining Solution: 5-10 minutes.
② Eosin Staining Solution: 3-5 minutes.
③ Rinse with tap water: 1-5 seconds.
3. Dehydration, Clearing, and Mounting
① 80% ethanol: 10-20 seconds.
② 90% ethanol: 10-20 seconds.
③ 95% ethanol: 2 applications, 1-2 minutes each.
④ Absolute ethanol: 2 applications, 2-3 minutes each.
⑤ Xylene clearing: 3 applications, 2-3 minutes each.
⑥ Mount sections with neutral balsam.
Staining Results: Cell nuclei appear blue; cytoplasm, muscle fibers, collagen fibers, etc., appear red in varying shades; keratin, red blood cells, etc., appear bright orange-red.
(2) Staining of Frozen Sections
1. Ethyl ether-ethanol mixed fixative for 5-10 seconds.
2. Rinse with tap water for 2-5 seconds.
3. Dropwise staining with Cole Hematoxylin Staining Solution for 1-2 minutes (may be heated to 50°C).
4. Rinse with tap water for 5-10 seconds.
5. Eosin Staining Solution for 2-5 seconds.
6. Rinse with tap water for 1-2 seconds.
7. 80% ethanol for 1-2 seconds.
8. 95% ethanol for 1-2 seconds.
9. Absolute ethanol for 2-5 seconds.
10. Phenol-xylene (1:3) for 2-5 seconds.
11. Xylene clearing for 3 applications, 2-5 seconds each.
12. Mount sections with neutral balsam.
Staining Results: Cell nuclei appear blue; cytoplasm and fibers appear red.
(3) Cell Staining
1. Fixation with 4% paraformaldehyde: 10-20 minutes.
2. Rinse with tap water: 2 applications, 2 minutes each.
3. Rinse with distilled water: 2 applications, 2 minutes each.
4. Follow the same staining, deparaffinization, clearing, and mounting steps as for paraffin sections, but reduce the reaction time accordingly.
Staining Results: Cell nuclei appear blue; cytoplasm and fibers appear red; keratin, red blood cells, etc., appear bright orange-red.
Precautions
1. Ensure thorough deparaffinization of sections. Replace serial ethanol solutions with fresh ones regularly.
2. Minimize the staining time for frozen sections.
3. Common bluing solutions include 0.2-1% ammonia water, Scott’s bluing solution, or 0.1-1% lithium carbonate solution.
4. For your safety and health, wear a lab coat and disposable gloves during operation.
5. This product is for scientific research use only. Any other uses are strictly prohibited.
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