Re: H & E Staining Method

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From:"Bryan Llewellyn" <>
To:"Histonet" <>
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

----- Original Message -----
From: Wenk, Lee & Peggy <>
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 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/ wrote:
> >
> > I am working with pig skin and doing H&E regressive staining using
> > 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
> > 10 minutes, which I think is too much, but this is what one of the
> > 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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