Re: H & E Staining Method
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From: | "Bryan Llewellyn" <bryand@netbistro.com> |
To: | "Histonet" <histonet@pathology.swmed.edu> |
Reply-To: | |
Date: | Sat, 21 Aug 1999 10:28:19 -0700 |
Content-Type: | text/plain; charset="Windows-1252" |
Sorry if you get two copies of this. I may have had a problem with the
address.
----- Original Message -----
From: Wenk, Lee & Peggy <lpwenk@netquest.com>
Subject: Re: H & E Staining Method
>What is non-acidified Harris hematoxylin?
Harris hematoxylin can be made with or without acetic acid. Without it is a
strongly staining non-selective and regressive formulation requiring
differentiation with acid alcohol, but demonstrating cement lines, mucin
etc. With acid it is more progressive (if timing is kept to 1 minute or
so), although I do not believe it can be compared to more traditional
progressive formulations. Acidified stain may not require differentiation,
although it often is a benefit. It also has a longer life due to resisting
alkali carry over, in contrast to most non-acidified formulations.
> Aren't all hematoxylins used in the H&E stain acidified, i.e.,
> pH'ed with acetic acid?
No. Most are, but some are not. Cole's formulation is a strongly staining
non-acidified formulation that gives results similar to acidified Harris. If
acid is added, it is a strongly staining (traditional) progressive stain.
>For our Mayer and Gill, we like the
> pH around 2.4-2.5. What is the best pH for Harris hematoxylin?
I don't think that adjusting pH to a specific number is really of much
benefit. Most formulas simply call for 20-50 mL per litre solution. Being
more precise than that seems pointless. Since the results differ depending
on whether it is acidified or not, the *best* pH would depend on what
results you want to get.
> Would there be a difference in pH if the ripening agent
> is mercuric oxide vs. sodium iodate?
Probably not. A solution with 50 g potassium or ammonium alum has a pH
around 5. The minor effect of oxidisers would likely be buffered out by the
amount of alum.
> Could this be part of the problem with overstaining? Too
> high of a pH, making is more non-specific - staining
> nucleoplasm and cytoplasm in addition to the chromatin
> material?
Probably it is the case. Non-acidified formulations do stain like this. In
addition, the long staining time is a factor. Harris usually calls for 3-5
minutes. Ten minutes is too long. A better choice would be Coles,
acidified or non-acidified depending on the results wanted. No
differentiation would be required. Staining time would be about 5 minutes.
Check stainsfile at http://members.pgonline.com/~bryand for a list of
formulas. Select staining methods, then mordanted hematoxylins. Select one
that is progressive with about 1.5-2.5 g dye per litre.
Bryan Llewellyn
>
> Edna_J_Gonzalez/Powderject@powderject.com wrote:
> >
> > I am working with pig skin and doing H&E regressive staining using
Harris
> > Hematoxylin (non-acidified). The problem is that the hematoxylin is too
> > dark and I can't differentiate what I need, especially it is too dark in
> > the basal layer and the epidermis of the skin. I need a H&E Regressive
> > Method that will be lighter. Currently I am staining with hematoxylin
for
> > 10 minutes, which I think is too much, but this is what one of the
methods
> > say. I know there are different H&E regressive methods (with different
> > times for Hematoxylin). Any suggestions?
> >
> > Edna Gonzalez
> > PowderJect Vaccines
>
>
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