[Histonet] Decalcification of mouse jaw and legs

idimitro at mun.ca idimitro at mun.ca
Tue Jul 14 09:25:33 CDT 2015

We do mouse legs routinely, either with Cal Ex II or EDTA. We look at the bone and the tissue around, so we have to have good fixation, decal., cutting and staining for both. 
Cal Ex II (formic acid) is a gentler decal agent than HCL (Plain "Cal EX") - and Cal Ex II keeps the H&E staining true - whereas HCL would make the Eosin dominate and stain everything!
For future - since Cal Ex II contains Formalin - on theory you can fix and decal at the same time, but I always do my fixation first and then decal after. Use chemical method to determine the end point.
Other ways to decal bone are with EDTA - I personally prefer it, though it takes a really long time, almost 2 months, it is better if you want to do IHC for certain markers that the acids might interfere with. We weight the samples before we start decal, and after that 2/week. The moment you stop seeing decrease in weight and see slight increase, that is the end point.
For cutting, I prefer samples decalc. with Cal Ex II, cuts without a problem.

Iliana Dimitrova, MLT, LHP, B.Tech., M.Sc. 

Histology Supervisor
Medical Education and Laboratory Support Services (MELSS)
Faculty of Medicine
Memorial University of Newfoundland
St. John's, NL Canada A1B 3V6

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-----Original Message-----
From: Swartwood, Steven J [mailto:Steven.Swartwood at cshs.org] 
Sent: July-10-15 7:23 PM
To: 'histonet at lists.utsouthwestern.edu'
Subject: [Histonet] Decalcification of mouse jaw and legs

Hello all,

I hope everyone is having or had a fantastic week,

I'm going to test a few different decal methods on mouse jaws and leg bones. I was wondering if anyone does this routinely out there who is willing to share a protocol. I've never done this on mouse jaw/leg bones before. I've only done this on human tissues. From what I've read it seems that for routine histology low % HCl is fine, but if I wanted to perform IHC then anywhere from 5-15% formic acid may be the best way to go. Nitric acid is probably too strong of a decal agent for these tiny specimens I would assume. Maybe an EDTA based decal agent may be best as well. I'm just really inexperienced with this and I'm very open to ideas and trying a few different methods.

Steven Swartwood HT(ASCP)
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