Hi, All,
I primarily section bone, and it's usually paraffin. I second the
vote on chilling the blocks (I chill mine in ice water instead of the
freezer, so if I forget my blocks the wax doesn't crack!), and using
ice water on a swab to keep the block chilled as you section.
Generally I section the entire bone, so about halfway through I need
to re-chill the block, but this whole chilling business may be
processing regimen- and wax type-dependent (uh, and the proficiency
of the user!). If you get a lot of compression of your ribbons, you
will get a lot of wrinkles on your slides, so optimize your cutting
for compression. If you look in the histonet archives, Rene Buesa
posted some time ago typical causes for ribbon/section compression- I
printed it out and keep it posted above the microtome to refer to
when I'm at a loss ;) ...
I transfer my sections to a room temp waterbath with some ethanol
before transferring to the heated waterbath. I also leave my sections
on the heated waterbath, usually 39-40C is a good temp for me, and
let them float until they look pretty much wrinkle-free by eye,
generally a few minutes. Having charged slides also helps, as the
cartilage in your section wants to stick to itself rather than a
slide, that's where a lot of the wrinkles are. A charged slide makes
it want to stick to the slide rather than itself a little more. Last,
after vertical air drying at room temp overnight (it's just
convenient for me, but a few hours should suffice), I "bake" my
slides at 45C overnight on a slide warmer, sometimes it's the
downstream processing that gives you the wrinkles, and good adhesion
helps, which is what the 'baking" is for. Keep in mind that I'm
self-taught, and a very mouse biology small lab, I do all the
processing, cutting, and staining myself. So, what works for me may
not be feasible for everyone, and may still not be the best way to do
things (although I do try to generate beautiful data).
Sincerely,
Nicole Collette
LLNL/ UC Berkeley
At 3:12 PM -0700 4/22/10, Joseph Saby wrote:
Brett-
Most wrinkles in decalcified bone sections come from stretching of
the decalified bone that occurs during the sectioning process. I
would suggest a rather simple solution. Allowing the sections to
flatten on the waterbath might take longer or a higher temperature.
If paraffin surrounding the bone section seems to be containing it,
not allowing it to expand to eliminate the wrinkles, gently tease it
off. After all, you want the bone section, not the paraffin. A
room temperature water bath (or 30% EtOH) to lay out the sections on
to tease out any wrinkles before transfering the sections to the
warm waterbath may also help.
I hope this helps!
Joe Saby, BA HT
________________________________
From: Adam . <[email protected]>
To: "Connolly, Brett M" <[email protected]>
Cc: [email protected]
Sent: Thu, April 22, 2010 11:12:26 AM
Subject: Re: [Histonet] need tips for cross-sectioning of cortical bone
Cutting bone is very hard, and I'm by no means an expert at it. Assuming the
blocks are properly fixed and decalcified, the best thing I've found is to
put the blocks at -20C for 5-10 mins to cool them, then right before you cut
them, rub a little ice water on the face of the block. That should help you
get some nice clean cuts. If the sections become hard to cut again, reapply
the ice water. If that stops working, back in the freezer they go.
Adam
On Thu, Apr 22, 2010 at 9:58 AM, Connolly, Brett M <[email protected]
wrote:
A colleague is having trouble getting wrinkle-free sections of
decalcified, paraffin embedded femur.
Any tips??
Thanks,
Brett M. Connolly, Ph.D.
Molecular Imaging Team Leader
Merck & Co., Inc.
PO Box 4, WP-44K
West Point, PA 19486
tel. 215-652-2501 fax. 215-993-6803
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