[Histonet] Overstaining - Mayers H&E

[Josephine Garcia]

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