[Histonet] immunocal

Gayle Callis gayle.callis <@t> bresnan.net
Thu Jan 3 16:19:51 CST 2008

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 

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
Bozeman MT

----- Original Message ----- 
From: "Michele Wich" <mwich <@t> 7thwavelabs.com>
To: <histonet <@t> lists.utsouthwestern.edu>
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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