Re: [Histonet] Decalcifying mouse heads

From:Gayle Callis

You did not say which antigens you are trying to stain for. 

For immunohistochemistry, we totally fix nasal turbinate portion of heads.
It is best to perfuse animal with a fixative via heart.  Animal is
anesthetized deeply so heart beats, and PBS is introduced via left
ventricle (hope I got that side correctly) to rinse out blood, then
fixative is injected via the same route. This is really not difficult to do.

We enjoy success with PLP fixative, suitable for immunohistochemistry.
After perfusion, the head (skin, muscles, brain, eyes, lower jaw with
tongue must be removed), then turbinates are immersed in PLP overnight with
one change of PLP (freshly made) for a second overnight immersion. Use at
least 20 times volume of fixative to size of sample, don't skimp on this.
We apply vacuum to suck air bubbles out turbinates to insure fixative good
contact with nasal mucosa.  

To decalcify, turbinates are immersed in 1.25% to 5% TETRASODIUM EDTA
dissolved in Dulbeccos PBS with pH adjusted to 7.6 with glacial acetic
acid.  Please note WHICH EDTA we use.  The head takes anywhere from 1 week
to 2 weeks in this decalcifier.  We cut off incisors and do a weight
loss/weight gain decalcification endpoint check UNLESS you have a FAXITRON
for xray endpoint check.  These heads are destined a prion and other prion
IHC, embedded in paraffin.  EDTA should be rinsed out with buffer - several

For frozen sections of turbinates, everything remains the same except
tetrasodium EDTA is 1.25% in DPBS containing 10% sucrose (I gave that
figure as 20% before, but 10% is the norm) until the head is decalcified.
Head is rinsed in 10% sucrose in DPBS, embedded in OCT, and snap frozen
using petri dish floating on layer of liquid nitrogen.  It cryosections

Decalcification in this EDTA solution is carried out at 4C, with daily
checks (very easy).  The sample is intially suspended in decalcifier and
vacuum is applied once again to pull any air out of turbinates.
Decalcification time is long, but success of IHC is alive and well. You can
either use a huge volume of decalcifier OR change it daily.  

If we only need routine H&E and no IHC on paraffin embedded turbinates,
then we decalcify in 10 - 15% formic acid, usually on NBF fixed bone (we
let heads fix longer, several days), do the same endpoint test or you can
use chemical test.  Head decalcifies in a couple of days, but is controlled
to avoid overexposure to acid.  For IHC, you may want to use buffered
formic acid (approx 4.5% formic with either sodium citrate or sodium
formate, easily made up in lab or commercial source) which is gentler and
touted for IHC.  EDTA is more protective of antigens, but some are robust
enough to withstand formic acid, and on occasion, HCl. I suggest you
determine the effects of your chosen decalcifier on your IHC staining
BEFORE you need option of IHC rather than get caught with decalcified bone
and no results at all.  For paraffin processing of turbinates, we do
extended schedule processing using Tissue Prep 2, a harder paraffin and
only 1 change of xylene, and 1 change of Clearite 3 or Propar to avoid
hardening bone excessively. 

We have done NBF fixed whole rat and mouse heads with lower jaws intact
using 10 - 15% formic acid with success, just rinse these in running water
for a hour and do extended processing.   

For some murine antibodies, fussy CD markers in particular, we frequently
do CryoJane tape transfer on  undecalcified bone to avoid all aldehyde

Whew, what a long lecture! 

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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