Combined staining with Hematoxylin and Eosin, referred to as HE staining for short, is the most basic staining method in routine pathological slide preparation and has extremely wide applications. Hematoxylin is a tawny crystal extracted from logwood, a plant native to Central and South America. It is a basic dye that forms hematein after oxidation. When used together with a mordant (commonly trivalent aluminum or iron salts), it can stain cell nuclei. In pathological diagnosis, teaching, and scientific research, HE staining is commonly used to observe the morphological structure of normal and diseased tissues. It can identify or differentiate certain abnormal substances and special components present in diseased tissues and cells. Special staining methods, enzyme histochemistry, immunohistochemistry, and other techniques are all performed on the basis of observing H&E-stained tissue sections. In H&E-stained sections, cell nuclei stain blue, and cytoplasm stains red, forming a distinct contrast that facilitates observation and analysis.
The Hematoxylin-Eosin (HE) Staining Kit (with Differentiating Solution) contains hematoxylin staining solution formulated with an independently developed recipe. The solution is composed of imported high-purity hematoxylin and oxidants, and is free of harmful substances such as mercuric oxide and methanol. It exhibits excellent staining effects on cell nuclei with the characteristics of being less prone to precipitation and metallic film formation. It has a broad application range, suitable for use in human, animal, livestock, and aquatic research fields, and can be applied to the staining of tissue paraffin sections, frozen sections, and tissue cells. Both hematoxylin staining solution and eosin staining solution are reusable.Note: This product is for research use only and not intended for clinical diagnosis or other purposes.
Staining Principles
1. Nuclear Staining Principle: Hematoxylin is a naturally occurring basic dye that can stain cell nuclei. The main component of chromatin in the nucleus is DNA. In the DNA double helix structure, the phosphate groups on the two nucleotide chains face outward, making the outer surface of the DNA double helix negatively charged (acidic). This allows it to easily bind to positively charged basic hematoxylin dye via ionic or hydrogen bonds, resulting in staining. Hematoxylin appears blue in alkaline solutions, so cell nuclei are stained blue.
2. Cytoplasmic Staining Principle: Eosin is a synthetic acidic dye that can stain cytoplasm under certain conditions. The main component of cytoplasm is protein, an amphoteric compound. Cytoplasmic staining is closely related to the pH of the staining solution. When the pH of the staining solution is below the isoelectric point of cytoplasmic proteins (4.7-5.0), cytoplasmic proteins undergo basic ionization and carry a positive charge, enabling them to be stained by negatively charged acidic dyes. Eosin dissociates into negatively charged anions in water, which bind to positively charged cations of cytoplasmic proteins, staining the cytoplasm red.
3. Differentiation Principle: After staining, the process of removing excess bound dye from tissues using specific solutions is called differentiation, and the solution used is called a differentiating solution. In HE staining, 0.5-1% hydrochloric acid alcohol is commonly used as the differentiating solution. Acids can destroy the quinone structure of hematoxylin, causing the dye to dissociate from tissues and fade. Most tissues must be differentiated with hydrochloric acid alcohol after hematoxylin staining to remove excess hematoxylin bound to cell nuclei and hematoxylin adsorbed by cytoplasm before eosin staining. This step ensures clear differentiation between nuclear and cytoplasmic staining.
4. Bluing Principle: After differentiation, hematoxylin exists in a red ionic state (appearing red) under acidic conditions and in a blue ionic state (appearing blue) under alkaline conditions. Tissue sections turn red or pink after differentiation with acidic alcohol. Immediately rinse the sections with water to remove acid and terminate differentiation, then treat them with weakly alkaline water to restore the blue color of hematoxylin-stained nuclei. This process is called bluing or regeneration of blue color. Alternatively, rinsing with tap water (especially warm water) can also achieve bluing, but it requires a longer time.
Materials to Be Prepared by User
1. Xylene or paraffin-embedded deparaffinization and clearing solution, graded ethanol, bluing solution, tap water or distilled water
2. Ether-ethanol mixed fixative, 4% paraformaldehyde, neutral balsam or environment-friendly mounting medium
Operating Procedures (For Reference Only)
(1) Staining of Paraffin Sections
1. Deparaffinization and Hydration of Sections
① Treat sections with xylene or paraffin-embedded deparaffinization and clearing solution twice, 5-10 minutes each time.
② (Optional) Treat with anhydrous ethanol twice, 3-5 minutes each time.
③ 95% ethanol: 3-5 minutes.
④ 90% ethanol: 3-5 minutes.
⑤ 80% ethanol: 3-5 minutes.
⑥ Rinse with tap water or distilled water (30-40℃ warm water is also acceptable): 1-3 minutes.
2. Staining
① Stain with hematoxylin staining solution: 3-8 minutes.
② Rinse with tap water or distilled water: 5-10 seconds.
③ Differentiate with acid alcohol: 2-5 seconds.
④ Rinse with tap water: 20-30 seconds.
⑤ Bluing with bluing solution or warm water: 20-40 seconds.
⑥ Rinse with tap water: 30-60 seconds.
⑦ Stain with aqueous eosin staining solution: 20-60 seconds.
⑧ Rinse with tap water: 30-60 seconds.
3. Dehydration, Clearing and Mounting
① 80% ethanol: 10-20 seconds.
② 90% ethanol: 10-20 seconds.
③ 95% ethanol twice, 1-2 minutes each time.
④ Anhydrous ethanol twice, 2-3 minutes each time.
⑤ Clear with xylene or paraffin-embedded deparaffinization and clearing solution three times, 2-3 minutes each time.
⑥ Mount with neutral balsam.
(2) Staining of Frozen Sections
1. Fix with ether-ethanol mixed fixative: 5-10 seconds.
2. Rinse with tap water: 2-5 seconds.
3. Stain with hematoxylin staining solution (dropwise application) for 1-5 minutes (heating to 50℃ is optional).
4. Rinse with tap water: 2-5 seconds.
5. Differentiate with acid alcohol: 2-5 seconds.
6. Rinse with tap water: 2-5 seconds.
7. Bluing with bluing solution or warm water: 2-5 seconds.
8. Rinse with tap water: 5-10 seconds.
9. Stain with aqueous eosin staining solution: 2-20 seconds.
10. Rinse with tap water: 5-10 seconds.
11. 80% ethanol: 1-2 seconds.
12. 95% ethanol: 1-2 seconds.
13. Anhydrous ethanol: 2-5 seconds.
14. Phenol-xylene (1:3): 2-5 seconds.
15. Clear with xylene or paraffin-embedded deparaffinization and clearing solution three times, 2-5 seconds each time.
16. Mount with neutral balsam.
(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, with appropriately shortened incubation times.
Staining Results
Cell Nuclei: Blue
Cytoplasm, Muscle Fibers, Collagen Fibers, Thyroid Colloid, etc.: Red of varying intensities
Keratin, Red Blood Cells, etc.: Bright Orange-Red
Precautions
1. Ensure thorough deparaffinization of sections. At low temperatures, sections can be treated in an incubator at 60-70℃.
2. Replace graded ethanol with fresh solutions regularly.
3. The duration of acid alcohol differentiation should be adjusted according to section thickness, tissue type, and reagent freshness. In addition, the tap water rinsing time after differentiation should be sufficient to completely remove residual 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 it in a sealed container.
5. Minimize staining time for frozen sections.
6. Bluing solution can be replaced with 0.2-1% ammonia water, Scott’s bluing solution, or 0.1-1% lithium carbonate solution.
7. For your safety and health, wear a lab coat and disposable gloves during operation.
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