[Histonet] cryosectioning calcified bone on manual freezing sledging microtome ?
In my research project I am using Differential Interference Contrast
(DIC) Microscopy to look at the attachment of the intervertebral disc to
the vertebral body. To date I have been fixing (Formalin) and
decalcifying (Formic Acid) the bone-disc-bone samples (approx 5mm by
10mm at the largest, of annulus and bone) before cutting 25-40micron
thick slices on a manual Freezing Sledging Microtome with disposable
steel blades (the sample is frozen onto a sample block with OCT using
For the next series of experiments I need fresh/hydrated slices of a
similar thickness that haven't been fixed or decalcified. I have access
to a tungsten carbide D profile microtome knife that I thought may work
(I have heard of other researchers cutting undecalcified bone set in
resin with tungsten-carbide blades). My attempts to date at using this
knife on the freezing sledging microtome have been unsuccessful.
I was wondering if;
A) the concept of cutting undecalcifed bone on a manual freezing
sledging microtome is fundamentally flawed (I've only been able to find
comments on cryo-sectioning undecalcifed bone on a cryostat). Is there
a different type of microtome that would be better and still allow me to
avoid both resins and decalcification ?
B) the blade could be blunt (I'm not sure of it's history - previous
user not the university anymore). It looks ok to me under the
microscope (but I'm no expert!). Should I be able to tell under a
I would appreciate any advise.
University of Auckland
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