Re: [Histonet] H and E staining


Few questions:

1. Directly from lithium carbonate to eosin?
There should be a running water rinse between. 2-5 minutes. Going from
bluing agent directly to eosin would change the pH of the eosin.

2. What's the pH of the eosin?
Should be around 4-4.5, acidified with acetic acid.

3. Eosin solvent?
70% alcohol works for us.

4. Water rinse after eosin?
Try going directly into 95% alcohol - 2 changes, 10 seconds with vigorous
dipping, then to 2-3 changes of abs. alcohol for the same, then xylene
several changes.

Peggy A. Wenk, HTL(ASCP)SLS
William Beaumont Hospital
Royal Oak, MI 48037

----- Original Message -----
From: "Ian Ward" 
Sent: Tuesday, December 07, 2004 4:24 AM
Subject: [Histonet] H and E staining

Hi all

I could use some advice, I have been trying to H and E stain some mouse
sections of the lungs, brain, spleen, liver, kidney, pancreas and heart. i
have used the following method:-
Xlylene     5minutes
Xlylene     5minutes
100% IMS 5minutes
100% IMS 5minutes
90% IMS  5minutes
70% IMS  5minutes
50% IMS  5minutes
running tap water   5minutes
Filtered Haematoxylin   5minutes
Quick wash in running tap water
Dip 20 times in Acid Alcohol
Quick wash in running tap water
Few seconds in Lithium Carbonate
1% Eosin   5minutes
Quick rince in running tap water
Take quickly though alcohols to xylene again

My problem is that for some reason very little of the Eosin can be seen on
the sections when I come to view them. Any ideas?



Ian Ward
Senior Technician
E Floor
The Medical School
Tel: 0115 9249924 Ext 36179

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