RE: [Histonet] Decalcification prior to Alizarine red or von Kossa possible?

From:Jack Ratliff



Alexandra,
 
It sounds like to me that you might be working with some sort of calcium phosphate material in the presence of demineralized bone and/or soft tissue in say a muscle pocket and looking for the presence/absence of bone formation. If you decal the tissue you will lose the ability to detect any clear mineralized bone as the alizarin red and Von Kossa reaction both identify mineralization (calcium). Also, if there are heavily mineralized nodules as you indicate, then I would say you do not have the option to cut as a paraffin block.
 
If I was doing this project, I would embed the tissue in methyl methacrylate (MMA), cut thin sections (4-6 microns) on a motorized rotary microtome with a tungsten-carbide knife, deplastify the tissue sections (similar to deparaffinization), and stain with the Von Kossa reaction and MacNeal's tetrachrome stain. This combination would then clearly yield the presence or absence of mineralization where the increasing degree of mineralization (dense older bone) would be seen as black and any newly formed mineralization (vital bone - osteoid) would be grayish-green. Furthermore, this staining combination will also allow for the identification of bone forming (osteoblasts/blue) and bone resorbing (osteoclasts/bluish-green) cells. In fact, this stain will clearly identify any active mineralization (osteoid) that has formed (osteoid) and show the active bone forming osteoblasts lining the surface as nice plump (single nuclei) cuboidal cells.
 
Additionally, if all you care about is mineralization or newly formed bone, you could also perform a Goldner's trichrome stain where any mineralization or newly formed bone (osteoid) will be highlighted as red. Hope this helps and let me know if you need any further assistance or advice.
 
Best Regards,
 
Jack



> Date: Thu, 9 Oct 2008 15:17:07 +0200> From: alexandra.meinl@gmail.com> To: histonet@lists.utsouthwestern.edu> Subject: [Histonet] Decalcification prior to Alizarine red or von Kossa possible?> > Hello all,> > I have to stain some specimens with bone formation in soft tissue. They want> me to do Alizarine red or Kossa stain.> But there are some heavily mineralized nodules in the tissue, I think they> can't be cut nicely without some kind of decalcification.> How should I treat these samples? Is there a way to decalcify them without> negative effects on the calcium stain?> > Thanks in advance!> > Alexandra> _______________________________________________> Histonet mailing list> Histonet@lists.utsouthwestern.edu> http://lists.utsouthwestern.edu/mailman/listinfo/histonet
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