Merissa, You should very easily be able to get a nice looking H&E from deplasticized undemineralized bone sections. In fact, try the protocol that I routinely use for my MMA embedded specimens:
Deplastification x 3 changes each for 15-30 minutes @ 55-60C w/ gentle dip and dunk at half way mark for each change 100% EtOH @ RT for 5 min 95% EtOH @ RT for 5 min 70% EtOH @ RT for 5 min dH2O @ RT for 5 min Hematoxylin 2 (from Richard Allen @ Fisher Sci) @ RT for 3 min Running Tap Water Rinse for 1 min Clarifier 2 (from Richard Allen @ Fisher Sci) @ RT for 20-30 seconds with a gentle dip and dunk at half way mark Running Tap Water Rinse for 1 min Bluing Solution (from Richard Allen @ Fisher Sci) @ RT for 20-30 seconds with a gentle dip and dunk at half way mark Running Tap Water Rinse for 1 min Eosin Y (from Richard Allen @ Fisher Sci) @ RT for 40 seconds 70% EtOH @ RT with 10 gentle dip and dunks 95% EtOH @ RT with 10 gentle dip and dunks 100% EtOH @ RT with 10 gentle dip and dunks 100% EtOH @ RT for 30 seconds Xylenes @ RT for 3 min Xylenes @ RT for 3 min Coverslip Your problem could be the type of hematoxylin you are using (progressive vs. regressive) and the length of time in solution. I would also recommend you try a Von Kossa with a MacNeal's tetrachrome counterstain, Goldner's trichrome, and maybe a toluidine blue stain with these type of sections. Best Regards, Jack Jack Ratliff Chairman, Hard Tissue Committee - National Society for Histotechnology On Mar 8, 2012, at 3:51 PM, "M.O." <modz9...@gmail.com> wrote: > Hello Histonetters! I am new to staining and can't seem to get good H&E > stains from deplacticized undecalcified bone with an implant. It seems as > though the hematoxylin is staining too dark. Any feedback would be much > appreciated! > > The protocol I am using is for after the deplasticization step and says: > > 100% EtOH for 3 mins, 70% EtOH for 3 mins > > Hematoxylin for 6mins, Rinse, .25% Acid Ethanol dip 8-10 times, Rinse > > 95% EtOH 1min > > Eosin for 2mins > > Quick Dip 95%, 95%, 100% 100% EtOH > > Soak in ProPar for 15+mins > > > Sincerely, > Merissa > _______________________________________________ > Histonet mailing list > Histonet@lists.utsouthwestern.edu > http://lists.utsouthwestern.edu/mailman/listinfo/histonet > _______________________________________________ Histonet mailing list Histonet@lists.utsouthwestern.edu http://lists.utsouthwestern.edu/mailman/listinfo/histonet