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Protocol: Hematoxylin Stain. [OHSU Intranet] [Search the OHSU Site] [OHSU Site Map] [Oregon Health Sciences University] [Neurological Sciences Institute] [Faculty] [Education] [Public] [Seminars] [Contact] [Directions] OHSU Website (NavBar): the Neurological Sciences Institute

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Hematoxylin Stain

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Hematoxylin Stain

Solutions

Hematoxylin

Hematoxylin 6.4g
Ammonium alum 60g
Ethanol 200ml
Glycerol 160ml
Distilled water 640ml

Mix for a day then allow the solution to ripen in the dark for at least a month before using.

Acidic Alcohol

Concentrated HCl 4ml
95% Ethanol 396ml

Bluing Agent

Sodium bicarbonate 1g
Distilled water 1 l

Staining

  1. Deparaffin and rehydrate slides.
  2. Slightly overstain the sections with hematoxylin 3-5 minutes.
  3. Remove excess stain in tap water for 2 minutes.
  4. Differentiate and restain a few seconds in acidic alcohol until sections look red.
  5. Rinse in tap water.
  6. Blue in bicarbonate until nuclei stand out sharply blue for 2 minutes.
  7. Rinse in running tap water.
  8. Dehydrate and clear, coverslip with Cytoseal.

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This document was created for the neurogenetics laboratory run by P. H. Reddy, Ph.D. in the NSI of OHSU. Page last updated: 18 Sept 2001.