[Histonet] HELP- Cryosectioning FAT!

[Balasubbramanian, Dakshnapriya]

Hi Laurie,

No, the tissue hasn't been fixed before freezing. My prof doesn't prefer the method, but if nothing else works, we might give it a try. 

Any suggested protocols for fixation?

Thanks!
Dakshna

----- Original Message -----
From: "Laurie J King" <king.laurie <@t> marshfieldclinic.org>
To: "Dakshnapriya Balasubbramanian" <dakshnapriya <@t> neo.tamu.edu>
Sent: Tuesday, May 20, 2014 10:37:35 AM
Subject: RE: [Histonet] HELP- Cryosectioning FAT!

Dakshna,

Has this tissue been fixed before freezing by any chance?

laurie

-----Original Message-----
From: histonet-bounces <@t> lists.utsouthwestern.edu [mailto:histonet-bounces <@t> lists.utsouthwestern.edu] On Behalf Of Balasubbramanian, Dakshnapriya
Sent: Tuesday, May 20, 2014 10:23 AM
To: histonet <@t> lists.utsouthwestern.edu
Subject: [Histonet] HELP- Cryosectioning FAT!

Hi all,

I've been having problems with cryosectioning fatty tissue for a long time now. The section leaves a hole in the middle. I know this is probably a much discussed topic, but I've tried a lot of strategies with no luck. The tissue is of mouse origin and has micro-lesions buried inside fat. So the fat needs to be cut in order to get to the lesion. I started with a temperature of -17C and tried upto -25C and also at -50C; didn't work at any of these temperatures. I tried rubbing the block, blade and anti-roll plate with dry ice-again,no luck. 

I rapid-freeze the tissue with OCT in LN2 and then store the block at -80C. Could I try sectioning the block directly from -80C? (never done that). 

Any other suggestions on how to tackle this?

Any advice is much appreciated!!

Thanks,
Dakshna

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