RE: cutting fixed "sucrose" livers
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From: | Cynthia Favara <cfavara@atlas.niaid.nih.gov> (by way of histonet) |
To: | histonet <histonet@magicnet.net> |
Reply-To: | |
Content-Type: | text/plain; charset="us-ascii" |
Irene,
I cut fixed cryopreserved [30% sucrose] mouse brain and
usually have to cut at a lower temperature than fresh frozen. In my limited
experience with this the temp is usually 10C cooler. We have two cryostats
and the temperature setting is different on each one. In essence I would
try colder and not be afraid to go to -30C.
I also have a higher incidence of sections floating off the slides
during staining[IHC]. I find that poly-l-lysine is the worst and super
frost the most satisfactory. Good luck!
Cynthia Favara
NIAID/RML
Hamilton, MT
-----Original Message-----
From: I.H.Straatsburg DIVG
[mailto:I.H.Straatsburg@AMC.UVA.NL]
Sent: Wednesday, January 13, 1999 12:17 PM
To: histonet@pathology.swmed.edu
Subject: cutting fixed "sucrose" livers
To start with: thank you for the suggestion to infuse fixed
tissue
with a sucrose gradient before cutting cryostat sections.
Without
sucrose, the morphology of the sections was awful.
Unfortunately, the
procedure I am using at the moment requires fixation of the
liver
tissue prior to freezing. I would rather do without.
Here comes the problem: the liver and kidney tissue infused
with 50%
sucrose in 0.1 M Phosphate buffer (until it sank) is very
difficult
to cut in a cryostat (-20 degrees Celsius; 8-10 micrometers
thickness).
Note: The tissue samples were not impregnated in OCT
compound (I take
that is 'Tissue Tec' or something similar) before freezing,
but
directly frozen either in liquid N2 or on a
cryo-boost-plate in the
cryostat cabinet.
Should I cut a lower temperatures? I have got the
impression, the
tissue is too soft, too sticky.
Is the impregnation with OCT compund essential?
I am very curious and thankful for any help!
Irene
===========================================================
Dr. I.H. Straatsburg
IWO1-155, Dept. Exp. Surg., Surgical Lab.,
AMC, Amsterdam, NL 1105 AZ
tel. +31.20.5666653
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