[Histonet] Frozen sectioning of pig skin

[Grantham, Andrea L - (algranth)]

Saro,
I'm certain there are differing thoughts on what to do about your tissue. 
Was the tissue fixed before it was frozen? How was it stored for the year it was in the freezer? Could be you have a piece of freeze dried pig skin that (if it cuts at all) will look awful.
Here is what I would do and I'll probably take some heat (no pun intended) on this but this is what I would do:
I'd drop the frozen chunk into fixative and let it thaw and then fix - time depends on the size of the chunk. After fixing I'd rinse the tissue, blot and drop into sucrose to cryoprotect. It is done when it sinks to the bottom of the sucrose. Blot. Then I would re-freeze in a frozen embedding medium like OCT or whatever you have - I like to put the tissue into some OCT and let it sit there for a while before freezing to absorb some of the OCT. I've had difficulty when there is sucrose solution on the outside of the tissue when it is frozen - makes it hard to get a decent section and there is much cursing going on in the lab.

Andi Grantham



On Mar 22, 2011, at 7:19 AM, Bascaramurty, Saro wrote:

> No, I did not. We are using Fisher's Thermo Shandon brand Cryomatrix. We will order this for next time and I will try and re-do the embedding after thawing it. Should I lightly rinse the block and pat-dry before re-embedding? 
> 
> Thank you,
> Saro  
> 
> -----Original Message-----
> From: Joel Israel [mailto:JoelI <@t> mcclainlab.com] 
> Sent: March 22, 2011 9:22 AM
> To: Bascaramurty, Saro
> Subject: RE: [Histonet] Frozen sectioning of pig skin
> 
> Did you thaw the tissue before you embedded in media?  If not, try it.
> I use NEG-50 from Richard-Allan Scientific to embed frozen tissue.  I have found it to be superior to others.
> 
> -----Original Message-----
> From: histonet-bounces <@t> lists.utsouthwestern.edu
> [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Bascaramurty, Saro
> Sent: Tuesday, March 22, 2011 10:00 AM
> To: histonet <@t> lists.utsouthwestern.edu
> Subject: [Histonet] Frozen sectioning of pig skin
> 
> 
> I have sectioned some formalin fixed, paraffin embedded pig skin blocks in the past, but never the fresh frozen pig skin blocks. The samples were frozen by the researcher in liquid nitrogen, but not covered in cryomatrix and have been stored in the deep freezer for over a year. I have now embedded those blocks in cryomatrix and wanted to prepare sections at 30um and 6-8um thickness. When I tried it, I had problems getting good quality sections in spite of using brand new blades. I tried lowering the temperature from -18 deg.C to -22 / -25 deg.C. I still couldn't get quality sections consistently. If anyone from histoland has some good tips to improve the quality of these sections, I would greatly appreciate it.
> 
> 
> Thank you,
> 
> Saro Bascaramurty
> 
> Technical Officer
> Institute for Biodiagnostics
> National Research Council
> 435 Ellice Avenue,
> Winnipeg, Manitoba. R3B 1Y6
> 
> Tel: 204-984-7166
> Fax:204-984-6978
> email:saro.bascaramurty <@t> nrc-cnrc.gc.ca
> 
> 
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