You can use any acid decalcifier for animal bone. If the Immunocal is
primarily a formic acid mixture, it will be slower than using a hydrochloric
acid decalcifier. Check the MSDS to see WHAT acid is being used. If you
want to ensure the bones are decalcified, then do an endpoint test to know
when the calcium is removed. If the bones are whole bones, then even goat
and/or sheep will be very large. Hopefully and to speed up fixation first
and then decalcification, the bones are reduced in size by cutting slabs or
open windows in the bone. 3 mm to 1 cm thick slabs will decalcify much
faster than a whole bone. One danger in working with whole bones or any
large animal bone is fixation must be total, or the acid decalcifier will
macerate the soft tissues and cells. This is a reason cutting open the
bones and/or letting them fix in NBF for a week or more is important. If
the bone is pink/reddish inside after you fix then slice open, then the
fixation should continue longer, and before you immerse in decalcifier.
The volume of decalcifier to bone we have always used is 20 to 1, and
suspend the bones in the decalcifying solution to allow the fluid to
surround all sample surfaces. If you do a chemical endpoint test, then
changing the decalcifying solution daily is important, although there is a
nice weight loss, weight gain endpoint test that we have used with great
success after we (stupidly!) gave away our FAXITRON X RAY unit. X ray is
the most sensitive endpoint determination, followed by chemical testing, and
even the weight gain/weight loss method, cheap, easy and fast to perform.
You only need a good balance that weighs in milligrams. I will be happy to
supply this method.
A duration will depend on the size of the bone and the type and
concentration of the acid one uses. Hence, endpoint testing takes away so
much of the guessing game on how long it takes to decalcify a bone since
age, size, and species of animals all are factors in how long it takes to
decalcify.
You can use 15% formic acid providing the bone is totally fixed, and another
good decalcifier is 4% hydrochloric acid/8% formic acid - one we have used
with great success for huge sheep distal femur slabs. Endpoint testing was
done daily, and we did not leave the bones in decalcifying solution over a
weekend IF the endpoint was close to completion. Overexposure aka over
decalcification is not a good thing for any bones, and staining will be
terrible if this happens.
Gayle M. Callis
HT/HTL/MT(ASCP)
Bozeman MT
----- Original Message -----
From: "Michele Wich"
To:
Sent: Thursday, January 03, 2008 1:27 PM
Subject: [Histonet] immunocal
Is anyone out there using Immunocal as a decalcifying agent for large
animal bones such as goat or sheep? If so, can you give me some idea as
to volume ratios, duration and the frequency at which the Immunocal must
be changed?
Thank you much!
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