Perfusion fixation is superior on this large an animal if you are able to
do it, a whole head takes a LONG time, plus many changes of fixative, not
good for preservation of bone and delicate structures, fixative can only
cross link so fast, NBF.  OR cut slabs, keeping orientation and sequence of
where slabs are taken, fix slabs, suspended in NBF. 
It does not hurt to cut the slabs on unfixed tissue, as long as blade
passes through the bone quickly and with water to keep saw blade from
burning bone and a minimum thickness of 3 mm, anything less  will pop bone
slab (potato chip syndrome) out of block when sectioning.  5 mm slabs work
nicely, too thick and you end up with processing problems and slow down
decalcification

How long depends on type of decalcifier - organic acid (formic), mineral
acid (hydrochloric, nitric) or chelator (EDTA) - (you did not indicate what
you are using), thickness of slab, type of bone, and what you want to do
with the bone afterwards,(IHC, routine staining) Endpoint determinations
would be an absolute must or you will NOT know WHEN the bone is completely
decalcified or to prevent overexposure to acid  aka "overdecalcification".
Chemical endpoint determinations are easiest, xray determinations are more
sensitive and much easier with large bones using a FAXITRON.  If you don't
do endpoint testing, you can ruin a specimen, been there, done that, a
faithful user of testing.    

Longer processing times are a must, automated with vacuum and pressure are
ideal, and up to 3 hours per station, could be longer or less depending on
overall size and thickness of slab.  How big is the dog? How big will the
slabs be?? 

Have decalcified a whole skinned rat head in less than a week but with
endpoint determinations to know exactly when calcium is gone, using 10 -
15% formic acid.  HCL and nitric would have decalcified faster than formic,
but do not care for damaging effects of stronger acids, even though formic
will and does damage nuclear staining, a given with acid decalcifiers
unfortunately.  You can minimize this with buffering (slower, formic acid
decalcifiers), and make sure you change the decalcifier daily unless you
are using a HUGE volume solution. 

Use a harder paraffin for both infiltration and embedding and improved
sectioning, Tissue Prep 2 (FISHER).

Whew, what a lecture!  TGIF!



     

  At 02:48 PM 1/19/01 -0600, you wrote:
>I am going to be working on a project where I need to get nose sections
>from a dog snout.  Does anyone out there in histoland have any experience
>with this kind of thing.  Would you suggest taking the sections first and
>then fixing and decalling, rather than fixing and decalling the entire
>head?  How long should decal take?  We have done rat turbinates, which we
>usually decal for a week, but the dog turbinate will obviously be a lot
>bigger and are made of much more dense bone.  Any suggestions or protocols
>would be much appreciated.
>
>Thanks
>
>Kris Eicher
>Toxicologist
>3M Pharmaceuticals
>St. Paul, MN
>
>
>
>
Gayle Callis
Veterinary Molecular Biology
Montana State University
Bozeman MT 59717-3610
406 994-4705
406 994-4303