Reagents
- Hematoxylin Powder: 3 grams
- Sodium or Potassium alum: 40 grams
- Mercuric Oxide: 0.5 grams
- Absolute ethanol: 20 ml
- Distilled water: 400 ml
Preparation of Stain
Mix the hematoxylin powder in ethanol and shake well. Heat the distilled water, add the alum to it and boil it well (at least for 5–10 minutes.). When the alum completely mixes with the water, add the hematoxylin solution and boil it again. After boiling, add mercuric oxide & boil it again.
Staining Procedure
1) Deparaffinize the slide by heating in hot incubation.
2) To remove paraffin, the slide has to be dipped in xylene for 1-2 minutes. Repeat the process four times.
3) Keep the slide in alcohol for 1-2 minutes to remove xylene. Repeat the process two times.
4) Wash the slide in running water.
5) Cover the slide with hematoxylin stain for 5 to10 minutes (according to maturation of stain).
6) Wash the slide in running water till the color appears blue. This is called bluing.
7) Decolorize the slide with 1% Acid-Alcohol, 1 dip. <decolourising/regressive stain>
8) Bluing the slide in running water until a violet colour appears.
9) Counter stain the slide with 1% aqueous eosin. Keep it for 1 to 2 minutes.
10) Wash the slide in water.
11) Allow the slide to dry.
12) Clear the slide with xylene and mount it with DPX or Canada Balsam.