[Histonet] Overstaining - Mayers H&E

Rene J Buesa rjbuesa <@t> yahoo.com
Mon Dec 7 11:24:04 CST 2009

You have to consider that the thicker the section the more intense the staining will be.
If you are using now 1 minute and they are still too dark, reduce the staining time even further.
Otherwise I don't see anything wrong with the rest of the protocol.
René J.

--- On Mon, 12/7/09, Josephine Garcia <jbdg <@t> u.washington.edu> wrote:

From: Josephine Garcia <jbdg <@t> u.washington.edu>
Subject: [Histonet] Overstaining - Mayers H&E
To: histonet <@t> lists.utsouthwestern.edu
Date: Monday, December 7, 2009, 11:42 AM

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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