[Histonet] Stain for osteoporotic bone in Spurr plastic

Fri Nov 9 09:28:44 CST 2012

Allison,

I had a great von kossa protocol for this but it was on glycol methacrylate
embedded bone, here it is, it uses the fluorescence property of eosin which
will stain the osteoid with the calcified bone covered by the silver von
kossa.

GMA is not removed
Rinse 5 micron sections in dih20, use clean glassware
Make 2% silver nitrate, put on the slides and expose them to uv (a lamp or
even in the window will do, for about 20 min.
The calcified bone should turn black
Rinse well in dih20, you can fix the silver with sodium thiosulfate but do
it quickly as it does remove some silver, rinse well in dih20
Hematoxylin nuclear stain, we used Gills#3 for 5 min., wash in tap water,
blue in ammonia water, wash well in tap water then to dih20
Make a 0.2% solution of aqueous eosin y, stain slides in this for 20 min.,
rinse in dih20 and airdry, coverslip with permount.
You can measure the calcified bone and nuclei in the light microscope, then
use a fluorescent scope with a fitc filter to see the osteoid, it will be
green just on the surface of the calcified bone which is black from the
silver.  We got a total sample area measurement, calcified area as a
percentage of total area, and osteoid area as a percentage of total area.
We did other measurements as well such as number of osteoclasts (we used a
Acid phosphotase (TRAP) enzyme histochemical stain for this) and number of
osteoblast (can use alkaline phosphotase enzyme histochemical stain for
these or we just counted them from the VK slide with the heme nuclear stain
by morphology, they will be lining the surface of the bone, very flat and
run together.)

Hope this helps.  Goldner TC is also used to measure osteoid but this worked
best for us.

Patsy Ruegg, HT(ASCP)QIHC
Ruegg IHC Consulting, LLC
40864 Arkansas Ave
Bennett, CO 80102
Phone: 303-644-4538
Fax: 720-859-4110
pruegg <@t> ihctech.net
-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Jack Ratliff
Sent: Thursday, November 08, 2012 4:52 PM
To: Malandra, Allison E; Histonet
Subject: RE: [Histonet] Stain for osteoporotic bone in Spurr plastic

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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