The combined staining of Hematoxylin and Eosin is commonly referred to as HE staining, which is the most widely 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 face outward, making the outer side of the DNA double helix negatively charged and acidic. This acidic property allows it to easily bind to the positively charged basic hematoxylin dye via ionic bonds or hydrogen bonds, thereby achieving staining. There are two main methods for the maturation of hematoxylin: natural oxidation and chemical oxidation. Natural oxidation refers to the exposure of hematoxylin to light and air; this process is relatively slow, taking approximately 3 to 6 months, but the resulting product maintains its staining ability for a long time. Ehrlich hematoxylin and Delafield hematoxylin are typical examples of hematoxylins matured by this method.
Delafield hematoxylin staining solution is highly stable and can be stored for 2 years in a sealed container after maturation. It stains nuclear chromatin clearly and delicately, though it requires a slightly longer staining time. It is suitable for staining sections used in teaching and scientific research, but it is not ideal for staining frozen sections.
Staining Principles
1. Principle of Nuclear Staining: 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 oriented outward, rendering the outer surface of the DNA double helix negatively charged and acidic. This acidity enables easy binding to the positively charged basic hematoxylin dye through ionic bonds or hydrogen bonds, resulting in staining. Hematoxylin appears blue in an alkaline solution, so cell nuclei are stained blue.
2. Principle of Cytoplasmic Staining: Eosin is a synthetic acidic dye that can stain cytoplasm under specific 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 undergo basic ionization, making the cytoplasm positively charged. Consequently, the cytoplasm can be stained by the negatively charged acidic dye (eosin). Eosin dissociates in water into negatively charged anions, which bind to the positively charged cations of cytoplasmic proteins, staining the cytoplasm red.
3. Differentiation: After staining, the process of removing excess dye bound to tissues using specific solutions is called differentiation, and the solution used is known as a differentiating solution. In HE staining, 1% hydrochloric acid-ethanol is commonly used as the differentiating solution. Acids can disrupt the quinone structure of hematoxylin, causing the separation of tissue and pigment and thus decolorization. For most tissues stained with hematoxylin, differentiation with 1% hydrochloric acid-ethanol is essential to remove excess hematoxylin bound to cell nuclei and hematoxylin adsorbed by cytoplasm. Only after this step can eosin staining be performed to ensure a clear distinction between the colors of cell nuclei and cytoplasm.
4. Bluing (Re-blushing): 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 ethanol. To stop the differentiation process, the acid on the tissue sections is immediately removed with water. Subsequently, weakly alkaline water is used to make the hematoxylin-stained cell nuclei appear blue—this process is called bluing or re-blushing. Additionally, rinsing with tap water can also achieve nuclear bluing, but it requires a longer time.
Materials to Be Prepared by the User
1. Hydrochloric acid-ethanol differentiating solution
2. Bluing solution (e.g., dilute ammonia water, lithium carbonate solution)
3. A series of ethanol solutions (for dehydration)
4. Eosin staining solution
5. 4% paraformaldehyde solution
Operating Procedures (for Reference Only)
(1) Staining of Paraffin Sections
1. Dewaxing of Sections to Water
① Xylene treatment: 2 times, 5-10 minutes each time.
② (Optional) Absolute ethanol treatment: 2 times, 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: 1-3 minutes.
2. Staining
① Stain with Delafield hematoxylin staining solution: 5-8 minutes.
② Rinse with tap water or distilled water: 5-10 seconds.
③ (Optional) Differentiation with hydrochloric acid-ethanol: 2-5 seconds.
④ Rinse with tap water: 20-30 seconds.
⑤ (Optional) Bluing with bluing solution: 20-40 seconds.
⑥ Rinse with tap water: 30-60 seconds.
⑦ Stain with eosin staining solution: 0.5-3 minutes.
⑧ 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 treatment: 2 times, 1-2 minutes each time.
④ Absolute ethanol treatment: 2 times, 2-3 minutes each time.
⑤ Clearing with xylene or paraffin-removing clearing solution: 3 times, 2-3 minutes each time.
⑥ Mount the section with neutral balsam.
(2) Cell Staining
① Fixation with 4% paraformaldehyde solution: 10-20 minutes.
② Rinse with tap water: 2 times, 2 minutes each time.
③ Rinse with distilled water: 2 times, 2 minutes each time.
④ Follow the same staining, dewaxing, clearing, and mounting steps as for paraffin sections, but reduce the treatment time accordingly.
Staining Results
Cell nuclei: Blue.
Cytoplasm, muscle fibers, and collagen fibers: Red (with varying shades).
Keratin, red blood cells, etc.: Bright orange-red.
Preautions
1. Ensure thorough dewaxing of sections. Replace the series of ethanol solutions with fresh ones regularly.
2. The differentiation time with hydrochloric acid-ethanol should be adjusted based on section thickness, tissue type, and the freshness of the staining solution. Additionally, the rinsing time with tap water after differentiation should be sufficient to completely remove residual acid.
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 use is strictly prohibited.
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