Veterinary Clinical Biochemistry and Laboratory Diagnosis-II

HAEMATOXYLIN AND EOSIN STAINING PROCEDURE

Ehrlich's Haematoxylin

• Haematoxylin - 2g
• Absolute alcohol - 100 ml
• Glycerin - 100ml
• Distilled water - 100 ml
• Glacial acetic acid - 10 ml
• Potassium alum approx - 15 g
• The haematoxylin is dissolved in the alcohol and the other chemicals are added.
• Natural ripening in sunlight takes about 2 months.
• It can be chemically ripened by adding sodium iodate using 50 mg for every gram haematoxylin but bench life is shortened.

Eosin Solution

• Eosin Y (water soluble) - 1 g
• Distilled water                - 80ml
• 95% alcohol                     - 320ml
• Glacial acetic acid           - 0.4ml
• Dissolve eosin in water and add 95 % alcohol. Add 0.4ml of acetic acid . The stain should be cloudy, add few more drops of acetic acid.

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Procedure

• Deparaffinise the section by xylol 5-10 minutes
• Remove xylol by absolute alcohol and hydration with down graded alcohols and water.
• Wash in tap water
• Stain with haematoxylin for 3-4 minutes
• Wash in tap water
• Allow the sections in tap water 5-10 mins
• Wash in tap water
• Counterstain with eosin 0.5% until section appear light pink (15 to 30 seconds)
• Wash in tap water
• Blot the section 
• Dehydrate with absolute alcohol
• Clear with xylol (15 to 30 seconds)
• Mount in Canada balsam or DPX Mountant
• Keep slides dry and remove air bubbles, if any.