Shanghai Yuanmu: What kinds of preparation methods are commonly used for HE staining reagents? -Huaqiang Electronic Network

Shanghai Yuanmu: What are the common preparation methods for HE staining reagents? I. Hematoxylin (1) Harris Hematoxylin Formula: Hematoxylin 1g, anhydrous ethanol 10ml, distilled water 200ml, potassium alum 20g, HgO 0.5g. Hematoxylin is first dissolved in absolute ethanol. Dissolve potassium alum in distilled water and heat to dissolve. Then add hematoxylin while stirring. Add a small amount initially to avoid boiling over. The heating process is critical here, as it affects the oxidation and dyeing effect. After adding all ingredients, cool the mixture quickly in ice water and let it stand overnight before filtering. Before use, add 5% glacial acetic acid. If stored longer, you may need to adjust the acetic acid slightly. The right amount of acetic acid ensures clear nuclear staining without background contamination. (2) Gill Modified Hematoxylin Solution Formula: Hematoxylin 2g, anhydrous ethanol 250ml, aluminum sulfate 17.6g, distilled water 750ml, sodium iodate 0.2g, glacial acetic acid 20ml. Dissolve hematoxylin in ethanol, then mix with aluminum sulfate solution. Add sodium iodate and glacial acetic acid. This semi-oxidized solution doesn’t require differentiation but benefits from it for better results. Sodium iodate’s quantity must be adjusted based on temperature. Too much can cause over-oxidation and poor staining. Accurate weighing is essential, so a balance with high precision is recommended. (3) Ehrlich Hematoxylin Solution Formula: Hematoxylin 2g, absolute ethanol 100ml, glycerol 100ml, glacial acetic acid 10ml, potassium alum 2-3g, distilled water 100ml. Mix hematoxylin in ethanol, then add glycerol and acetic acid. Dissolve potassium alum in water and combine with the hematoxylin solution. Let it sit in sunlight for about two months until it turns brown. This method yields a stable solution that can be used for long-term staining. (4) Mayer Modified Hematoxylin Solution Formula: Hematoxylin 2g, anhydrous ethanol 40ml, potassium alum 100g, distilled water 600ml, sodium iodate 0.4g, glacial acetic acid 20ml. Dissolve potassium alum in water and gently heat. Mix with hematoxylin solution in ethanol, add sodium iodate, and ensure full dissolution. This progressive solution also requires careful handling like the Gill version. II. Eosin (1) Water-Soluble Eosin Formula: Eosin (water-soluble) 2.5g, distilled water 500ml. Dissolve eosin in water, add concentrated hydrochloric acid, let it stand, filter, and dry. Prepare a saturated solution in 95% ethanol before use. Adding a small amount of glacial acetic acid improves the staining effect. (2) Alcohol-Soluble Eosin Formula: Eosin (alcohol-soluble) 2.5g, 95% ethanol 1000ml. Add a few drops of glacial acetic acid to enhance staining. Adjust pH to 4.5 using NaOH or acetic acid for optimal cytoplasmic staining. III. Hydrochloric Alcohol Differentiation Solution Formula: Concentrated hydrochloric acid 0.5–1ml, 75% ethanol 99ml. This solution helps differentiate stained tissues by removing excess hematoxylin. IV. Cleaning Solution Soak in 30% hydrochloric acid or 5% phenol solution for 30 hours, then rinse thoroughly. Useful for cleaning slides or coverslips. V. Equivalent Hydrochloric Acid To prepare 1N HCl in 1000ml, how many milliliters of concentrated HCl are needed? The exact volume depends on the concentration of the stock solution, typically around 8.3 ml of 37% concentrated HCl.