[Histonet] Stain for osteoporotic bone in Spurr plastic

[Jack Ratliff]

Allison,
I am not exactly certain if this protocol will work for Spurr's embedded specimens, but here is the protocol that I use for deplasticized sections of methyl methacrylate embedded undemineralized bone (non-decalcified):
Xylenes - warmed @ 50 C for 30 min w/ dip & dunk @ 15 min interval
Xylenes - warmed @ 50 C for 30 min w/ dip & dunk @ 15 min interval
Xylenes - warmed @ 50 C for 30 min w/ dip & dunk @ 15 min interval
100% EtOH - room temp for 5 min
95% EtOH - room temp for 5 min
70% EtOH - room temp for 5 min
DI H2O - room temp for 5 min
Silver Nitrate (20 g) + DI H2O (400 ml) - room temp (in dark protected from light) for 5 min
DI H2O rinse - room temp (protected from light) for 1 min
DI H2O rinse - room temp (protected from light) for 1 min
DI H2O rinse - room temp (protected from light) for 1 min
Sodium Carbonate (22.5 G) + DI H2O (337.5 ml) + 37% Formaldehyde (112.5 ml) - room temp (protected from light) for 2 min
DI H2O rinse - room temp for 1 min
DI H2O rinse - room temp for 1 min
Sodium Thiosulfate (40 g) + Potassium Ferricyanide (2 g) + DI H2O (420 ml) Solution - room temp for 30 sec(solution stable for 30-60 min after addition of potassium ferricyanide)
Running Tap Water Rinse - 10 min
Counterstain w/ MacNeal's Tetrachrome (12 g) + DI H2O (600 ml) - room temp for 6-8 min(stir continuously w/ heat @ 60C for 48 hours covered, then filter with paper towel)
DI H2O rinse - room temp for 1 min
DI H2O rinse - room temp for 1 min
DI H2O rinse - room temp for 1 min
70% EtOH - room temp for 1 min
100% EtOH - room temp for 1 min
Xylenes - room temp for 3 min
Xylenes - room temp for 3 min
Coverslip w/ Eukitt


Hope this helps! Please feel free to contact me if you have any questions or concerns.
Best Regards,
Jack

Jack L RatliffOwner, Ratliff Histology Consultants, LLCChairman, Hard Tissue Committee - National Society for Histotechnology
(317) 281-1975jratliff <@t> ratliffhistology.com LinkedIn Profile:  http://www.linkedin.com/profile/edit?trk=hb_tab_pro_top




> From: allison-malandra <@t> uiowa.edu> To: histonet <@t> lists.utsouthwestern.edu
> Date: Thu, 8 Nov 2012 22:52:42 +0000
> Subject: [Histonet] Stain for osteoporotic bone in Spurr plastic
> 
> Hi there -
> 
>   We are trying to stain osteoporotic bone that was embedded using Spurr plastic.  We need to be able to differentiate osteoid from mineralized bone.  We have tried Gio's trichrome and Goldner's trichrome with no success, both surface staining and on deplasticized slides.  We are thinking of trying Von Kossa, does anyone have a recipe/protocol for this?  Or other types of stains that you have had success with?  Thank you!
> 
> Allison Malandra, DVM
> University of Iowa, Bone Healing Research Lab
> 
> 
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