RE: [Histonet] Cryostat sectioning of mammary gland

From:"Monfils, Paul"

The problem with sectioning mammary gland is, in a word, FAT.  Fat doesn't
freeze at -20 degrees.  It also doesn't freeze very well at -35 degrees. A
mammary gland where fibrosis or tumor has replaced a lot of the fat cuts
quite well.  But typical mammary gland does not. First, you will need to get
the block even colder, by using a freon spray. This can bring the block
temperature down to about -50 degrees. If you are using an anti-roll plate,
spray that first, generously, and wipe off any excess refrigerant. Then
spray the block generously for a few seconds, then IMMEDIATELY cut your
section, before the block can warm at all.  Also, it may not be possible to
cut 4 or 5 micron sections.  A 10-15 micron setting will give you a much
better chance of getting a section of fatty tissue. Cutting the sections
faster also frequently helps with tissue of this type.  Freeze the block
with the spray, then turn the handle quickly, so that you "chop" the section
off the block (so to speak) rather than "slice" it off.  Finally, it usually
isn't the fat that you are interested in, when sectioning this kind of
tissue.  You may find that even though there are holes where some of the
larger fat masses were, the stroma is still intact in the section.

> ----------
> From: on behalf of Tessa
> Murray
> Sent: 	Wednesday, August 10, 2005 1:50 PM
> To:
> Subject: 	[Histonet] Cryostat sectioning of mammary gland
>    I'm looking for some advice on cryosections.  We are trying to collect
>    frozen  sections of mammary gland tissue and are experiencing problems
>    - we do a lot of paraffin work here so the cryostat technique is quite
>    new to us.  We find that the knife cuts the OCT fine, but when it hits
>    tissue  it does not cut so we get sections with tissue shaped holes in
>    them.   We've  tried  changing the block and knife temp (we have tried
>    altering  them  between  -30  to -35C) but nothing seems to help.  Any
>    advice appreciated...
>    tess
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