[Histonet] Cutting undecalcified bone

[Patsy Ruegg]

I have experience cutting frozen sections of whole rat tibias without decal
(John Tarpley and I did this work together) which in my experience could
only be done using the tape transfer technique.  I use a D profile tungsten
carbide knife(the bevel is only on one side unlike a C profile which has the
same bevel on both sides, this is like the old triangle shaped glass knives)
this knife requires a pretty straight cutting angle compared to the C
profile which is more angled, thus the reference Liz had about me changing
the knife angle.  I have tried everything from heavily coated 6X to the
lightest 1/2X coated slides.  I have still to find a technique which will
give me perfect sections of bone everytime.  The problem for me is not
getting the perfect section cut but getting it attached to the coated slide
without bubbles under the tape and then removing the tape without leaving
some of the section on the tape.  This can be very frustrating and expensive
to cut a beautiful section only to have it pull off with the tape, the
expensive slide is lost and your beautiful section is useless.  
This is not only the case for undecalcified bone, I have been having the
same old problem with cornea implants.  Some things to try include:  try to
cut a thinner section like 4 microns (if your knife is really sharp this is
not a problem), the thinner section will lay flatter on the coated slide.  I
do expose the UV for 2-3 times with about 10 secs. Between or the fuse will
blow.  Pull the tape off diagnally from corner to corner very slowly and
smoothly.  When appling the tape with the section to the coated slide "role
the hell out of it" to borrow a phrase from Gayle, to make sure it gets
really well attached to the slide before UV.  I have had some luck with
preparing the slide, exposing it to UV and then placing it on dryice for
5-10 min. before removing the tape, but again I never get every section in
good shape and actually lately I have been getting more bad sections than
good, which is driving me crazy.  There are people up at CSU using this to
cut horse carpal bone, I can give you their contact info if you like.  I
think I did better with bone than I am doing with cornea right now.
Oh, for bone I try to cut as cold as possible, about -35 I have been cutting
the softer tissue warmer -18---24, hey maybe that's my problem I should go
back to the cold cold bone method, it can't hurt at this point.
I snap freeze samples by surrounding them with OCT and either slowly
lowering into liq. Nitrogen or isopentane and dryice.
Patsy
  

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu
[mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Stephen
Peters M.D.
Sent: Thursday, June 02, 2005 10:18 AM
To: Histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] Cutting undecalcified bone

Nancy,
 
I have experience cutting frozens on undecacified bone specimens for routine
surg path cases using conventional technique but have no experience with the
tape transfer system.
 I routinely use disposible low profile blades which in not optimum for
bone. I imagine a stronger high profile blade or knife will work better. My
success has varied with the hardness of the bone. Trebecular bone coming
from older patients can cut fairly easily porvided the blade is sharp, and
the section is taken with a fluid continuous motion. The result is a
surprisingly intact section. The harder the bone ( typically the younger the
patient ) there will be more damage to the blade in each pass.  The sections
will be streaked and splitting requiring one or more blade changes by time I
get a resonable section. It is quite a juggling act.
Cutting hard cortical bone is often very frustrating and very difficult to
get any resonable section.  From what you describe as bubbles, I am guessing
you are getting sections of bone thicker than you are hoping for and as a
result when you roll it out, the thicker bone pieces are protecting the rest
of the section from rolling. Often the first section of any tissue will be
considerably thicker than those that follow after a few fluid turns of the
wheel. If you are not letting a few pass your first section may be
considerably thicker and as a result will shatter more and create thicker
trabeculae which may be leading to your problem.
 The equivalent using conventional technique will give me a section that my
coverslip will not  lie flat against the slide and my mounting medium will
not spread.
I look foreward to other peoples experience on this subject.  
 
Stephen
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