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" <[email protected]>
To: <[email protected]>
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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