[Histonet] need tips for cross-sectioning of cortical bone

[Nicole Collette]

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 . <anonwums1 <@t> gmail.com>
>To: "Connolly, Brett M" <brett_connolly <@t> merck.com>
>Cc: histonet <@t> lists.utsouthwestern.edu
>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 <brett_connolly <@t> merck.com
>>  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
>  > brett_connolly <@t> merck.com
>>
>>
>>
>>  Notice:  This e-mail message, together with any attachments, contains
>>  information of Merck & Co., Inc. (One Merck Drive, Whitehouse Station, New
>>  Jersey, USA 08889), and/or its affiliates Direct contact information for
>>  affiliates is available at http://*www.*merck.com/contact/contacts.html)
>>  that may be confidential, proprietary copyrighted and/or legally privileged.
>>  It is intended solely for the use of the individual or entity named on this
>>  message. If you are not the intended recipient, and have received this
>>  message in error, please notify us immediately by reply e-mail and then
>>  delete it from your system.
>>  _______________________________________________
>>  Histonet mailing list
>>  Histonet <@t> lists.utsouthwestern.edu
>>  http://*lists.utsouthwestern.edu/mailman/listinfo/histonet
>>
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://*lists.utsouthwestern.edu/mailman/listinfo/histonet
>
>
>
>      
>_______________________________________________
>Histonet mailing list
>Histonet <@t> lists.utsouthwestern.edu
>http://*lists.utsouthwestern.edu/mailman/listinfo/histonet