[Histonet] Cutting undecalcified bone

Instrumedics info <@t> instrumedics.com
Mon Jun 6 11:34:39 CDT 2005

I know from experience that many bone labs with the CryoJane system have 
difficulty in getting  "perfect" sections and transferring  them fully 
intact. However, many of them seem to find a technique for success.

Dr. Rowe and his colleagues at UConn have been very successful with 
tape-transfer in cutting decalcified and undecalcified bone. I believe he 
has given a workshop(s) on his method.

Perhaps you can contact Dr. Rowe's lab.


----- Original Message ----- 
From: "Patsy Ruegg" <pruegg <@t> ihctech.net>
To: "'Stephen Peters M.D.'" <petepath <@t> yahoo.com>; 
<Histonet <@t> lists.utsouthwestern.edu>
Sent: Friday, June 03, 2005 5:38 PM
Subject: RE: [Histonet] Cutting undecalcified bone

>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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