RE: IF's and skin

From:"Gutierrez, Juan"

Hi Deb!  I once did an FDA project in which I had to do immunos on over 500
frozen skin biopsies.  I refroze each and everyone of them with no ill
effects on staining.  My protocol was as follows:
1. Thaw skin in cold PBS.
2. Pat tissue dry with filter paper.  Just enough to remove excess wetness.
3. I used an intermediate size cryomold with a drop of OCT in it.
4. Orient your biopsy in the mold and fill up the rest with OCT.
5. Float mold in isopentane that's been cooled to at least -50c.
Do not use the alcohol in dry ice method to freeze for it will make the OCT
"gummy" and you will not be able to get sections.
And if at all possible, remove the fat from the sample(a lot easier to cut).
Juan C. Gutierrez, HT(ASCP) 
Histology Supervisor 
Christus Santa Rosa Hospital 

-----Original Message-----
From: []
Sent: Thursday, June 05, 2003 5:57 PM
Subject: IF's and skin

1) Does anyone know if skin that is misoriented, freeze artifact....any
other problems once frozen can be thawed and refrozen again?   

2) Would thawing/refreeze affect the IF staining afterwards?   

3) If a skin has freeze artifact and if it can be thawed, does it need to be
rolled in talc powder before refreezing?  (Muscle biopsies that are
thawed/refrozen are rolled in talc powder to get rid of freeze artifact.) 

I'm hoping for some quick, good advice before I address a problem at work. 
Thanks to whomever can help. 

Deb King, HT (ASCP) 

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