Reagents
- Harris hematoxylin or Weigert’s iron hematoxylin
- 1% acid-fuchsin solution
- Saturated picric acid
- 1% Acid-alcohol for Harris hematoxylin only
Prepare fresh picric acid-fuchsin solution before staining. To prepare the solution, take 9 drops of saturated picric acid in a test tube. Add 1 drop of 1% acid fuchsin solution (1 in 10 dilution).
Weigert’s Iron haematoxylin:
a) Haematoxylin: 1 gram and absolute alcohol: 100 ml. Prepare a solution (solution: A).
b) 30% FeCl: 4 ml, concentrated HCl: 1 ml, and distilled water: 95 ml. Mix to prepare a solution (solution: B).
Mix both solutions (solutions A and B) in equal volume before staining. Always use a freshly prepared solution.
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 and wait for 5–10 minutes.
6) Just dip the slide in 1% acid-alcohol solution. This step is not required for iron hematoxylin).
7) Wash the slide in running tap water.
8) Flood the slide with picric-acid fuchsin solution for 1 minute and 30 seconds.
9) Blot the slide rapidly and let it dry. Xylene and mount.