Re: [Histonet] Freezing Biopsies

From:Gayle Callis


We experienced this too.  We solved by using Tissue Tek plastic embedding 
molds 10 x 10 mm size.  Cut excess edges for a better heat sink around 
embedding well except for a small tab to hold onto.   Carefully pick a mold 
that is not deformed already - look for the flat ones, not rounded!  Tissue 
Tek has newer round molds that do not require trimming, very nice, tidy and 
Unfortunately, other brands of plastic molds we tried had thicker plastic 
compared to Tissue Tek molds and blocks twisted out at freezing.

Coat bottom of mold with a THIN film of OCT.  Biopsy is laid out so it is 
as flat as possible in OCT film on bottom.  Carefully, slowly - add more 
OCT down corner of mold or away from top of biopsy to keep it from floating 
up.  Overfilling with OCT results in cracked blocks; fill to top of well.

  We tilt the beaker with cold isopentane , and introduce mold so bottom 
makes contact with isopentant first.  When OCT starts to turn white ON 
BOTTOM,  immerse fully -about 10 sec or so.  Bottom freezing first anchors 
biopsy and keeps it flat, no lift up syndrome.  If frozen block face has 
any deformity to it caused by a funky mold - just smear a thin layer of OCT 
over block face to fill in these depressions, etc.  to have a flatter block 
face before trimming.   Thin layer is important as we tried to avoid any 
excess warming of block once it is frozen solid.

Have fun!!

At 07:07 PM 1/25/2005, you wrote:
>         I'm snap freezing kidney biopsies in OCT using isopentane that is 
> chilled with liquid nitrogen. I'm having a problem where the area that 
> the biopsy is gets raised up above the surface of the block. I'm not sure 
> what is going wrong and more importantly what to do about it. Has anyone 
> had similar problems and how did you solve it?
>Amos Brooks
>Histonet mailing list

Gayle Callis
Research Histopathology Supervisor
Veterinary Molecular Biology
Montana State University - Bozeman
PO Box 173610
Bozeman MT 59717-3610
406 994-6367 (lab with voice mail)
406 994-4303 (FAX)

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