[Histonet] mouse leg decalcification

Gayle Callis gayle.callis at bresnan.net
Sun Jul 19 11:57:52 CDT 2015



The advice Iliana gave was excellent.   We avoided HCl decalcifiers for any
bone decalcification due to the damaging effects on staining (H&E, Giemsa)
but more importantly on antigens if you plan on immunostaining.    You did
NOT say what you what kind of staining you wanted  with murine jaws and leg
bones?   Please enlighten us about what you need post decalcification.   


Buffered formic acid i.e. Cal Ex II is a good choice but with any acid
decalcifier and there are other commercial buffered formic acid reagents
available so check the MSDS to see how they are made.   You should do a
decalcifcation endpoint check to not over expose the tissues/cells, etc to
acids, even this gentler buffered formic acid.    


My EDTA decalcification method does not take two months to be complete and I
will be happy to send you this method from a publication by Dr. Webb Jee
many years ago.   This is  14% tetra sodium EDTA, pH 7.6 dissolved in
Dulbeccos PBS or distilled water.   This particular EDTA is very alkaline(pH
11) when dissolved and the pH must be adjusted down using acetic acid, not
HCl.   Very alkaline EDTA from pH  8  to pH 11 can potentially damage
alkaline sensitive protein linkages so we stay with the pH commonly used
with TRIS Buffered saline, making final decalcified bone safe for
immunostaining.   EDTA decalcification acts as a function of pH,  so that
decalcification is slower at pH 7 than at pH 7.6.   This is a chemical fact
explained in text books.  


We do endpoint checks faithfully for any and all  bone decalcification.  The
most sensitive methods are with an xray (Faxitron) or Micro CT units if you
have these available.   The weight loss/weight gain method is easy, cheap
and but you need a balance that weighs in mg units, preferably three places.
I have used the weight gain/weight loss method for years and did it daily
with all acid decalcifying agents, but you can do it less often with EDTA.
Bones were immersed in formic acid decalcify solution in late afternoon and
do the test the next morning since some leg bones will almost be totally
done by the end of the next day.   We suspend bones in nylon biopsy bags and
stir or rock the container of decalcifying solution during decalcification
to release the CO2 bubbles  collecting on the bone surface which then causes
bones to float upwards.   CO2 is given off as part of the acid
decalcification chemical process.      


We make sure the bones are totally fixed for 4 days or longer to protect
from the effects of acid i.e. bad nuclear staining, damage to antigens,
BEFORE ever going into any decalcifying agent.   We normally do a 10% - 15%
formic acid made up from 88 - 90% stock formic acid in the lab although
buying commercial buffered formic acid.  Buffered formic acids are
approximately 4.5% formic acid with either sodium formate or sodium citrate
buffering salts.   They can take a bit longer but murine bones aren't very


We have done whole mouse and whole rat skulls in the past with perfect H&E
staining after using total NBF fixation and 10% formic acid decalcification
using endpoint checks daily.      


The key is to NBF fix totally, do endpoint check to ensure removal of
calcium and use either a formic acid decalcifying solution.    


Good luck


Gayle Callis



You wrote:   


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


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


-----Original Message-----

From: Swartwood, Steven J [mailto:Steven.Swartwood at cshs.org
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> ] 

Sent: July-10-15 7:23 PM

To: 'histonet at lists.utsouthwestern.edu
<http://lists.utsouthwestern.edu/mailman/listinfo/histonet> '

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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