Staining with hematoxylin and eosin-xylol
Primary Fixation
Fix specimens or tissues and affix them to slides or coverslips. AFA is the preferred fixative. We move our coverslips from columbia jars to ceramic staining racks for processing. All stains and solutions are kept in screw top jars. We remove the lids as needed and move the racks from jar to jar and then tighten down the lids for storage.
Fixative Rinse
Rinse out excess fixative in 70% Ethanol for 3 minutes. Specimens that have been stored in 70% ethanol can go directly to staining.
Hydration
Harris hematoxylin is water based, so specimens have to be hydrated before staining. Hydrate by graded alcohol series 50%, 3 min; Water, 5 - 7 min.
Staining
Harris Hematoxylin is a regressive stain, that is, specimens are overstained and then destained to remove excess stain and differentiate structures. Stain in Harris Hematoxylin for 15 minutes.
Destaining and differentiation
Rinse out excess stain in gently running tap water for 3 minutes and proceed to destaining. Destaining and differentiation is the critical step that determines the quality of the final preparation. Destain in 1% acid alcohol until the cytoplasm has only a faint stain but the sharp nuclear stain still remains. Briefly rinse in tap water to remove excess acid and halt destain.
Bluing and setting hematoxylin
Dip in ammonia water or lithium carbonate water until specimens are bright blue (3-5 dips)and rinse in gently running tap water for 10 - 20 minutes. Hematoxylin is pH sensitive shifting from red under acidic conditions to blue under alkaline conditions. The red state is unstable and fades over time or when exposed to light while the blue state is time and light fast. Thus the bluing step sets the hematoxylin and ensures stain longevity in the final preparation.
Dehydration
Dehydrate in a graded ethanol series: 50%, 70%, 95% (3 min each); 100% (3 changes, 3 min each).
Clearing and counterstaining
Clear in a graded xylene series: 50% xylene/50% Absolute alcohol (3 min); Zelmer's Eosin-Xylol (3 min); 100% xylene (2 changes, 3 min each).
Mounting
Mount in warm (37-40 C) Damar Balsam. (Note: Canada Balsam tends to become acidic as it polymerizes and will color-shift and fade hematoxylin stains over time.)