[Histonet] acid alcohol versus acetic acid rinse, calling on Gary Gill to comment

Gayle Callis gcallis <@t> montana.edu
Tue Oct 12 15:09:27 CDT 2004

Robyn and others,

You wrote:

After the hematoxylin and then the water, are you dipping them in 1% acid 
alcohol a couple of times to decolorize them?

One needs to be careful here.  When one says they use acid alcohol, they 
need to say what hematoxylin (name please) they use i.e. either progressive 
or regressive methods to avoid confusion.   The only time we use 1% 
hydrochloric acid/alcohol mixture is after Harris's hematoxylin and then to 
"differentiate" or remove some of the hematoxylin stain from the nuclei to 
make it crisp and delicate, i.e.a regressive hematoxylin stain protocol.  I 
have no doubt it could be used with progressive, but have rarely seen this 
done with concern this stronger acid could remove too much of the 
progressive type hematoxylin. Hopefully Gary Gill will comment on this.

With progressive hematoxylins, Gill (half oxidized hematoxylin)  including 
Richard Allan and other vendors formulations available, one does not 
require "differentiation" with an acid alcohol as these hematoxylins are 
not designed (hmm correct term?) to "overstain" the nuclei.   We have 
always used a mild acid rinse aka clarifying rinse with 4% acetic acid to 
remove background from the slide surface with any progressive hematoxylin 

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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