Hi HistoNetters

The original Sorvall procedure for H & E staining of JB-4 sections used
Harris' hematoxylin.  This is the protocol:

Harris' Hematoxylin and Eosin
SOLUTIONS: 

Harris' Hematoxylin
2.5g    Hematoxylin crystals
25.0ml    Ethyl alcohol, 100%
50.0g    Ammonium or potassium alum
500.0ml    Distilled water
1.25g    Mercuric oxide (red)

Dissolve the hematoxylin in the alcohol, and the alum in the water by the
aid of heat. Remove each from heat and mix the two solutions. Bring to a
boil as rapidly as possible. (Limit this heat to less than 1 minute and stir
often.) Remove from heat and add the mercuric oxide slowly. Reheat to a
simmer until it becomes dark purple; then remove from heat immediately and
plunge the vessel into a basin of cold water until cool. The stain is ready
for use as soon as it cools. Addition of 2-4ml of glacial acetic acid per
100ml of solution increases the precision of the nuclear stain. Filter
before use.
Acid alcohol
1000.0ml    Ethyl alcohol, 70%
10.0ml    Hydrochloric acid, concentrated
Ammonia Water
1000.0ml    Tap water
2-3ml    Ammonium hydroxide, concentrated
Eosin-Phloxine Solution
Stock Eosin
1.0g    Eosin Y, water soluble
100.0ml    Distilled water
Stock Phloxine
1.0g    Phloxine B 
100.0ml    Distilled water
Working Solution 
100.0ml    Stock eosin
10.0ml    Stock phloxine
780.0ml    Ethyl alcohol, 95%
4.0ml    Glacial acetic acid

Make up working solution as needed. Working solution should be changed at
least once a week.

STAINING PROCEDURE:


1.    If sections are Zenker-fixed, remove the mercuric chloride crystals
with iodine and clear with sodium thiosulphate (hypo).
2.    Stain in Harris' hematoxylin for 10 minutes.
3.    Rinse in tap water.
4.    Differentiate in acid alcohol for 2-3 quick dips.
5.    Wash in tap water.
6.    Dip in ammonia water until sections are bright blue; 2-3 dips.
7.    Wash in running tap water for 10-20 minutes.
8.    Stain with eosin-phloxine for 2 to 5 minutes at room temperature or
1minute at 60ºC. For even staining results, dip slides several times before
allowing them to set in the eosin for the desired time.
9.    Dip in 95% alcohol until excess eosin is removed; 3-5 dips.

Blow dry.
Mount.

RESULTS: 

Nuclei -- blue with some methachromasia; cytoplasm -- various shades of pink
identifying different tissue components.

best regards,
Steven Slap

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Marketing Manager/Microwave Product Specialist
Hacker Instruments & Industries, Inc.
http://www.hackerinstruments.com
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