Reagents and Preparation of Stain
Solution A
- Acid fuchsin: 0.5 gram
- Glacial acetic acid: 0.5 ml
- Distilled Water: 100 ml
Solution B
- Phosphomolybdic acid or oxalic acid: 1 gram
- Distilled water: 100 ml
Solution C
- Methyl Blue or light green: 2.0 gram
- Glacial acetic Acid: 2.5 ml
- Distilled water: 100 ml
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) Remove mercury pigment by iodine thiosalfate.
6) Wash the slide in tap water.
7) Stain nuclei by hematoxylin (preferably iron hematoxylin).
8) Differentiate in acid-alcohol.
9) Wash well in tap water.
10) Stain with solution A for 5 minutes.
11) Rinse with distilled water.
12) Treat with solution B for 5 minutes.
13) Drain the excess solution.
14) Stain with solution C for 2–3 minutes.
15) Rinse with distilled water.
16) Treat with 1% acetic acid for 2 minutes.
17) Dehydrate through graded alcohol
18) Clear in xylene & mount.
Results
Muscle: Red
Nuclei: Blue
Fibrous Tissue: Green