76 8 Haematoxylin and Eosin Stain of the Tissue Section

Steps: No. 2:
Ferric chloride: 10 g
• Haematoxylin is dissolved completely in Distilled water: 100 ml
absolute alcohol. No. 3:
• Add distilled water. Iodine: 1 g
• Keep the solution for 4–6 weeks for ripening. Potassium iodide: 2 g
Distilled water: 100 ml
Solution 2 No. 4: Working solution
Ferric ammonium sulphate: 5 g Add:
Distilled water: 100 ml No. 1: 40 ml
Step: Dissolve violet crystal of ferric ammo- No. 2: 16 ml
nium sulphate in distilled water. No. 3: 16 ml
Staining Mix those solutions in order.
Steps:
• Dewax the tissue.
• Absolute alcohol. • Dewax.
• 95% ethyl alcohol. • Serial grade of alcohol for hydration.
• Keep section in mordant solution (solution 2) • Stain by freshly prepared working haematox-
for 60 min. ylin solution for 10 min.
• Rinse in distilled water. • Rinse in water.
• Stain by solution 1 (Heidenhain’s haematoxy- • Differentiation: by 2% ferric chloride.
lin 0.5%) for 60 min. • Wash by tap water.
• Rinse in water. • Remove iodine by 95% ethyl alcohol: 5 min.
• Differentiate in 5% alum solution. • Counterstain: 1% eosin for 1–2 min.
• Wash in running water: 5–10 min. • Dehydrate.
• Dehydrate. • Clean by xylene.
• Clean by xylene. • Mount by DPX.
• Mount in DPX.

Result Elastic bre takes black stain.

Result Target tissue takes black stain.

8.6.3 Tungsten Haematoxylin

8.6.2 Verhoe ’s Iron Haematoxylin
Tungsten haematoxylin is used to demonstrate nerve
Verhoeff’s iron haematoxylin is used to stain tissue (such as astrocyte), muscle tissue and colla-
elastic tissue. The stain provides very good con- gen. Here phosphotungstic acid is used as mordant.
trast for microphotography. Ferric chloride is
used here as mordant. Strong iodine is used for 8.6.3.1 Preparation
oxidizing agent. The whole solution should be Solution A
prepared just before use because the solution Haematein: 0.8 g
may be over oxidized if kept more than 1 h. Distilled water: 1 ml
Step: Dissolve 0.8 g haematein in 1 ml dis-
8.6.2.1 Preparation tilled water.
No. 1: Solution B
Haematoxylin: 5 g Phosphotungstic acid: 0.9 g
Alcohol (100%): 100 ml Distilled water: 9 ml