[Histonet] Optimizing H&E Stains for undecalcified bone

[Jack Ratliff]

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." <modz9636 <@t> 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
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