[Histonet] Overstaining - Mayers H&E

[Bryan Llewellyn]

There doesn't appear to be anything wrong with the staining schedule.

Mayer published more than one alum hematoxylin and one of them is a strong 
regressive type, and would produce the staining you see.  Is it possible 
that has been used instead of his progressive one?  If it has, make the 
following diluent.

Potassium or ammonium alum 50 g
Citric acid 1 g or glacial acetic acid 20 mL
Distilled water 1L

Then dilute the strong alum hematoxylin 1 part with 3 parts diluent.

Or,

Immediately before the 80% ethanol prior to the eosin, place into a solution 
of 0.5% acid ethanol for a few seconds.  Wash well to blue, and carry on.

If that doesn't correct it, then make up a fresh batch of Mayer's hemalum.

Bryan Llewellyn



----- Original Message ----- 
From: "Josephine Garcia" <jbdg <@t> u.washington.edu>
To: <histonet <@t> lists.utsouthwestern.edu>
Sent: Monday, December 07, 2009 8:42 AM
Subject: [Histonet] Overstaining - Mayers H&E


> Hi all,
>
> My (frozen-section, fixed) slides are coming out much too dark 
> (overstained
> purple) and I'm not sure why. They are 15-20 micrometer slices of rat
> gastrocnemius muscle. Can someone please look over our current protocol 
> and
> tell me what I'm doing wrong? Thanks! Here it is:
>
> 1. Perfuse animal with 4% paraformaldehyde fixative.
> 2. Soak in 0.4% paraformaldehyde
> 3. Sucrose cycle (5% rinse, 10%, 20%, 30% soak)
> 4. Embed in OCT, Frozen sections (15-20 micrometers)
> 5. Let dry for 15-30 min
> 6. Stain as follows:
>
> - Distilled H2O (quick dip)
> - Mayer's Hematoxylin - 1min (originally we were dipping for 5-10 minutes. 
> I
> slowly reduced the time to 2min, then 1min, then 30s... still 
> overstained!)
> - Running lukewarm tap water - drain and continuously fill - 15min or 
> until
> water runs clear
> - Distilled H2O (quick dip)
> - 80% EtOH - 1-2min
> - Eosin - 2 min
> - 95% EtOH I - 10sec
> - 95% EtOH  II - 10sec
> - 100% EtOH I - 10sec
> - 100% EtOH II - 10sec
> - Xylene I - 2min
> - Xylene II - 2min
>
> 7. Coverslip and let dry overnight
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