NALT/bone undecalcified frozen sections

From:Gayle Callis <>

      Kim Kusser <>


I would like to cut mouse skulls (sans lower jaw, and front incissors)
without decalcifing them first. Has anyone out there done something like
this, or have an idea of how it could be accomplished?The purpose of this
is to stain NALT (nasal associated lymphoid tissue). This generally
involves several lymphoid markers (CD3, Thy1.2, IgD, B220, CD21,
CD11C...basically anything we can think of : ). BTW, I also multiple label
my stuff. 2 for chromogens, and 3 for fluorescence. Hence, the reason why I
prefer frozen. 

Thanks in advance


If you do not need to see nasal turbinates at all, there is a very slick
way to dissect out NALT (nasal associated lymphoid tissue - in human this
is analogous to tonsils).  Otherwise, using the Instrumedics Cryojane is
the only successful way for undecalcified bone frozens on this area of
murine anatomy.  The NALT is located above the 3 - 4th palate ridges, with
teeth below it and all the tiny swirling turbinate bone before, above with
skull behind it. Mouse bone anatomy doesn't cooperate on working with this

You need to have a bone saw that will not tear up delicate bones associated
with the NALT, Buehler Isomet is very nice if you remove incisors.  We have
not found a way to remove molars just below NALT without damaging it.
Pulling mouse teeth tears up everything.

If you do frozens, use a tungsten carbide knife, as sharp as possble, to do
the sectioning. It is best to cut through the teeth to the NALT (had the
best sections this way) This dulls the TC knife rapidly, so more than one
is needed as back up. This gives a full length view of NALT, however, you
can cut turbinates from the top down into the NALT.  This also sacrifices
valuable knife edge, but bone is not as hard as teeth. Researcher here
bought Cryojane specifically for working with NALT in situ, undecalcified!
Looking at $7000 to start with.  Special snap freezing is in order to not
split the delicate turbinates.  

We prefer to do dissection to eliminate all bone, snap freeze with NALT
facing up, connective tissue against mold, on a flat dry ice block with OCT
in a tiny Tissue Tek Cryomold to keep NALT as flat as possible.  The palate
connective tissue curls and messes up NALT if you do other snap freezing
technic.  We cut serial frozen sections,  Normal NALT, uninflamed -
anywhere from 25 - 30 serial, and inflamed NALT as many as 80 serial
sections. Have learned to view NALT unstained or you can lose it all in a
few thick cuts. 

I strongly suggest doing the dissection, as bone frozens are very expensive
to deal with in terms of time, morphology, and mainly equipment.  

Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
Bozeman MT 59717-3610

406 994-6367
404 994-4303 (FAX)

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