Practical methods for cartilage

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From:Gayle Callis <>
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>I've noticed several messages in the archive about decalcifying cartilage,
>but I would also like to know how decalcifying affects the non-calcified

Acid decalcifiers tend to hydrolyze the proteins in the nuclei, and alter
the staining characteristics.  People tend to have weak and sometime NO
staining of nuclei (result of overexposure to acid a.k.a.
"overdecalcification")with the hematoxylin stain.  
EDTA will extract proteoglycans from the cartilage matrix but not be a
problem for what you want, that is of more concern when staining for
specific proteoglycans via IHC or special staining (SafO/Fast Green and
Toluidine blue for evaluating articular cartilage). 

I would like to compare the shapes of chondrocytes from the
>various zones of fresh cartilage, and I need the cell shape to be
>preserved a well as possible.  

Does anyone have a preferred method to
>process fresh cartilage, down to the calcified zone, that they can send to
The original article by Rosenberg on using Safranin O/Fast green was done
on frozen sections of cartilage.  Cartilage contains a goodly amount of
water, and this is removed by processing through alcohols, resulting in
cells that may not retain their true shape with a shrinking rate of around
25%! Maybe paraffin is not the best method due to this.  Can honestly say I
have never seen articular cartilage or even growth plate cartilage
chondrocytes unaffected by paraffin processing. 

One could try Glycol methacrylate on cartilage, takes small, thin pieces,
and process these without alcohol, using GMA/water gradients instead of
alcohol gradient that remove water.

OR snap freeze a piece of cartilage and cryosection with a tungsten carbide
knife to get through the calcified areas (one could try a regular blade
down to calcified area) fix with NBF for 5 - 10 minutes, rinse, decalcify
the frozen section with EDTA (Patsy Ruegg uses post frozen section
decalcification), and stain with toluidine blue or even an H&E. 

Keep us posted on how your project turns out, very interesting technical

Preferred stains to visualize cell shape?  Any help will be greatly
>Jerry Hu
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303

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