Hematoxylin and Eosin staining, commonly referred to as HE staining, 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 nature allows it to easily bind to the positively charged basic hematoxylin dye via ionic bonds or hydrogen bonds, thereby achieving staining.
Carazzi Hematoxylin Staining Solution is mainly composed of hematoxylin, potassium aluminum sulfate, etc. It belongs to a type of alum hematoxylin solution. This staining solution contains a small amount of hematoxylin and does not form an oxide film. It stains cell nuclei very clearly without coloring cytoplasm or fibrous components, and is classified as a progressive stain. It is particularly suitable for counterstaining cell nuclei after staining techniques such as enzyme histochemistry and immunohistochemistry, especially when acid treatment cannot be applied after special staining. In such cases, the staining time is relatively short (usually 8-10 minutes), and bluing can be performed immediately after staining without the need for differentiation.
Staining Principle
1. Principle of Cell Nucleus 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 Cytoplasm 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 clear distinction between the colors of cell nuclei and cytoplasm.
4. Bluing (or Re-blushing): 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 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.
Operating Procedures (for Reference Only)
1. Adjust the staining operation and the amount of stain used according to the specific experimental requirements and the type of tissues or cells being stained.
2. Hydrochloric acid-ethanol differentiation is not required. The staining time is generally 8-10 minutes; for regressive staining, it takes 15-30 minutes, and for progressive staining, 8-15 minutes. Typically, controlling the time at 10 minutes is sufficient.
Precautions
1. Ensure that sections are thoroughly dewaxed.
2. Replace the series of ethanol solutions with fresh ones regularly.
3. Minimize the staining time for frozen sections as much as possible.
4. Common bluing solutions include 0.2-1% ammonia water, Scott’s bluing solution, or 0.1-1% lithium carbonate solution.
5. For your safety and health, wear a lab coat and disposable gloves during operation.
6. This product is for scientific research use only and must not be used for other purposes.
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