[Histonet] Re: Decalcification of Rat Hind paws

From:Gayle Callis

We are doing a similar study with mouse paws, difference being larger rat
paws, yours will need longer fixation, and you can speed up decalcification
and not damage the cartilage by using either of the two following
decalcifiers.  Make them up in house.  Both decalcifiers do not damage
proteoglycans, and work beautifully with SafO/FG staining, also T blue. 

Fast:  8% formic acid/4% hydrochloric acid, change daily, do endpoint
determinations as pure estimation of time CAN get you in trouble with
under- and over removal of calcium. There is a way to do it without going
to a lot of work, OR use xray methods with Faxitron for the most sensitive
detection.  You can also do chemical testing, but we are finding the weight
loss/weight gain method works fine.  You will probably have paws done in a
couple of days this decalcifier, but I wouldn't bet on it via estimation.
Paws take longer than you think - the arrangement of tiny bones so close to
each other tends to slow down fixation AND decalcification.  Slit open open
skin on back of paws to help out too if you can get this done.   

Faster than your buffered formic acid:  15% formic acid, do endpoint
determinations and change daily but decalcification will take longer than
the above combo acid decalcifying solution. 

Once you do the endpoint determination on a few of the paws (they are all
about the same size, age, etc) you have an estimate of time for the rest.
I will be happy to send the quick method for doing this. 

I have found paws need to have extended processing due to the same tissue
density/boney problems. 

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology 
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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