Re: Mineralised Bone biopsies

From:Gayle Callis

You can make up your own polymethylmethacrylate (PMMA, and preferred by
many for bone, microtomed sections where PMMA can be removed for
immunostaining, ie Neil Hand protocols).  You need tungsten carbide tipped
blades, d profile, there are c profile available, d is excellent and a
microtome that will be strong enough to do the cutting, Olympus has one (do
not recall model) Leica 2155 and 2165.  As for a good sledge for
mineralized bone in PMMA, a Polycut, but it is pricey as are all
microtomes/knives for these protocols. 

It is cheaper to not use kits, and you can use PMMA methods where washing
inhibitor away is avoided, getting rid of tedious, messy, handling of toxic
substance is reduced.  You will need a fume hood vented to outside, a
refrigerator, waterbath for polymerization.  Not sure if Technovits has a
PMMA kit suitable for microtoming, if so, it would be worth a try. 

You can use an automated processor for dehydration and clearing, but not
infiltration with PMMA, use to vacuum dessicators for hands on infiltration
with PMMA.  The polymerization is best done in a waterbath for even
heating, ovens do not provide even heating with this step. 

There are many publications in J of Histotechnology concerning these
methods, including Hand's antigen retrieval, look for these authors, Diane
Sterchi, J. Eurell, Cathy Sanderson (now Mayton), Chappell (hope I spelled
that correctly). 

Working with undecalcified bone samples is more time consuming, there are
no STAT protocols, or you end up with a mess of bubbly, polymerized goo. 


At 03:37 PM 2/27/02 +1100, you wrote:
>Hi Histonetters,
>We are in the process of setting up a Mineralised Bone Histo section in our
>Department and we need some advice. We are continuing the work of another
>department who have handed over this job to us and would like to streamline
>the process a bit. The way they make up their resin is long, tedious and
>tricky, so that is the first thing I would like to "fix". Secondly we use
>"spare time" on another departments' old sledge microtome to cut the bones
>and I would like to purchase our own equipment to get away from having to
>fit in with another lab.
>So firstly, has anyone had any experience cutting Mineralised (not
>decalcified!) bone on a heavy duty rotary microtome or should we be using a
>Secondly, what resins do you use and are they available in easy to use kit
>Thanks for any help in advance
>Andrew Kennedy
>Senior Science Officer in charge - Histopathology.
>Department of Anatomical Pathology
>Concord Repatriation General Hospital
>Hospital Road
>Concord, NSW, Australia
>Phone: +612 9767 6115
>Fax: +612 9767 8427
>"Noli illegitimi carborundum"
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>Central Sydney Area Health Service.
Gayle Callis
Histopathology Supervisor
Veterinary Molecular Biology - Marsh Lab
Montana State University - Bozeman
19th and Lincoln St
Bozeman MT 59717-3610

406 994-6367
406 994-4303 (FAX)

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