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

From:Tony Henwood

Is the precipitate alum?
Old solutions often leave a precipitate similar to fine blue tissue paper
(the alum component). 
Haematein dye precipitate appears as dark blue granular dust on the
Usually a filter of the haematoxylin solution will solve this. 
You will probably find that if your haematoxylin is too old the nuclei will
begin to appear brown.


Tony Henwood JP, BAppSc, GradDipSysAnalys, CT(ASC)
Laboratory Manager
The Children's Hospital at Westmead,
Locked Bag 4001, Westmead, 2145, AUSTRALIA.
Tel: 612 9845 3306
Fax: 612 9845 3318

-----Original Message-----
From: Gayle Callis [] 
Sent: Wednesday, 13 October 2004 6:09 AM
To: Robyn Vazquez;
Subject: [Histonet] acid alcohol versus acetic acid rinse, calling on Gary
Gill to comment

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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