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 _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet