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 it. Add the hematoxylin solution while stirring slowly to prevent boiling over. This process requires careful control of heat and timing. After mixing, cool the solution rapidly in ice water and let it sit overnight before filtering. Before use, add 5% glacial acetic acid. The amount of acetic acid affects the clarity and staining quality—too little leads to poor contrast, too much may reduce nuclear staining. (2) Gill Modified Hematoxylin 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 does not require differentiation, but proper dyeing improves results. Sodium iodate quantity should be adjusted based on temperature—higher in colder seasons. Accuracy in measurement is crucial to avoid issues like poor staining or co-staining. (3) Ehrlich Hematoxylin 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, combine with the hematoxylin solution, and let it sit in sunlight for about two months. When the color turns brown, it’s ready for use. This method offers a stable, long-lasting stain. (4) Mayer Modified Hematoxylin Formula: Hematoxylin 2g, anhydrous ethanol 40ml, potassium alum 100g, distilled water 600ml, sodium iodate 0.4g, glacial acetic acid 20ml Heat potassium alum in water, dissolve hematoxylin in ethanol, mix both solutions, and add sodium iodate. This progressive solution also benefits from proper dyeing techniques. As with the Gill formula, precise measurements and environmental adjustments are essential. 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 overnight, filter, and dry. Prepare a saturated solution in 95% ethanol and dilute before use. Add a small amount of glacial acetic acid for better performance. (2) Alcohol-Soluble Eosin Formula: Eosin (alcohol-soluble) 2.5g, 95% ethanol 1000ml Add a few drops of glacial acetic acid to enhance staining. Modern red dyes are often too acidic, so adjust pH to around 4.5 using NaOH or acetic acid for optimal cytoplasm staining. III. Hydrochloric Alcohol Differentiation Solution Formula: Concentrated hydrochloric acid 0.5–1ml, 75% ethanol 99ml Use this solution to differentiate stained sections for better contrast. IV. Cleaning Solution Soak in 30% hydrochloric acid or 5% phenol solution for 30 hours, then rinse thoroughly. This is useful for cleaning slides or cover slips. V. Equivalent Hydrochloric Acid To prepare 1N HCl in 1000ml, you need approximately 83.3ml of concentrated HCl. Always handle with care and follow safety protocols. This detailed guide helps ensure accurate and consistent HE staining results in histology labs.

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