Merissa,
You should very easily be able to get a nice looking HE 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 HE
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
