Re: [Histonet] H and E staining

From:Gayle Callis

You do not need to do a water rinse AFTER the eosin unless you are working 
with aqueous eosin.  This rinse is taking water soluble eosin out of the 
tissue.  Stain in eosin, then go to 95% alcohol X 2 changes, 100% X 2, 
etc.   The 95% alcohol will differentiate the eosin without removing it 

We stain murine FFPE tissues with eosin (alcoholic) for 1 minute and have 
often backed off that time if the sections are too red, a common, annoying 

Hopefully, you do a good 1 minute running tap water rinse after Lithium 
carbonate to remove this alkaline solution.  IF you alter the pH of eosin 
with this alkaline solution i.e. cation carryover, the eosin will not have 
a correct  pH to do its job in pH 4.5 range.

By quick washes,  1 minute is quick for us, we can go longer but NEVER less 
than that.  Adequate rinsing makes sure hematoxylin, acid and alkaline 
solutions are rinsed away thoroughly.

We add as 70% alcohol rinse before the eosin to do the following:

1) remove any residual cation carryover from LiCO3 - even after it's water 
2) equilibrate the section to the same concentration of alcohol as the 
eosin is dissolved in.

If you need any guidelines, go to Richard Allan website staining manual and 
read about H&E  staining - it is free and excellent how one can manipulate 
this stain to suit their needs i.e more contrast, deeper staining, etc, etc.

At 02:24 AM 12/7/2004, you wrote:
>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
>NG7 2UH
>Tel: 0115 9249924 Ext 36179
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Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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