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

From:"Bryan Hewlett"

Gayle and others,

With either acid, 1% acid alcohol or 4% aqueous acetic acid, what you are
doing is "acid differentiation"!
The use of terms such as 'Clarifier" is nothing but obfuscation!!!!!!
It doesn't matter if the hematoxylin solution is used progressively or
regressively, acid rinses will still do two things;
a) change the colour to red (hematoxylin is an indicator after all)
and b) destabilize the dye lake attachment to the chromatin and
progressively remove it.
The degree of both things happening is related to the pH and the time of

I must also point out that a couple of dips in 1% acid alcohol will not
"decolourize" the section.
Decolourization is the complete removal or loss of colour(see Oxford
The use of the term decolourize should be restricted to these occasions and
not used to describe differentiation.

OK, rant over.

Irascible old guy,


----- Original Message -----
From: "Gayle Callis" 
To: "Robyn Vazquez" ; 
Sent: Tuesday, October 12, 2004 4:09 PM
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
> or regressive methods to avoid confusion.   The only time we use 1%
> hydrochloric acid/alcohol mixture is after Harris's hematoxylin and then
> "differentiate" or remove some of the hematoxylin stain from the nuclei to
> make it crisp and delicate, i.e.a regressive hematoxylin stain protocol.
> 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
> formulation.
> 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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