Re: [Histonet] h&e staining on frozen sections
This is the technique that I have found to be useful.
Prepare your tissue as usual.
Section on the cryostat and handle the sections for fluorescence, ISH etc as
Cut the frozen section to be stained for H&E at 4-6 micrometer thickness,
lift the anti-roll plate and pickup the flat section on a room temperature,
positively charged slide.
Immediately immerse the slide in a coplin jar containing the following
ETOH.... 70.0 mL
Formalin (37-40% formaldehyde).... 10.0 mL
Glacial acetic acid........5.0 mL
D.Water to make up to 100 mL.
Allow the section to fix for 2 - 5 minutes
Place slide in 70% ETOH x2 for 30 seconds each, followed by two changes D.
Proceed with the H&E stain.
The resulting stained section will be virtually indistinguishable from a
FFPE stained section.
----- Original Message -----
From: "Charles Murtaugh"
Sent: Saturday, April 30, 2005 12:03 AM
Subject: [Histonet] h&e staining on frozen sections
I have a problem that has plagued me for years, and is particularly
annoying right now because I am trying to move entirely away from doing wax
sections to frozen sections (for better preservation of antigens, ease of
processing, etc.). I study mouse developmental biology, and I always like
to stain one section from every block I process by standard H&E techniques.
On wax sections, these are always nice, but on frozen they are invariably
For frozen sections, I first fix the tissues 1-4 hrs in 4% PFA/PBS, then
treat overnight with 30% sucrose/PBS and embed in OCT. My students and I
usually get really nice frozen sections, from 8-12 microns depending on what
we want to look at, and typically we analyze by fluorescent microscopy or in
situ hybridization. Whenever we do H&E staining, though, we get all this
artifactual shrinking of the tissue, presumably due to the subsequent
dehydration, as well as very poor differentiation of hematoxylin-stained
nuclei. With wax neither of these is a problem, even though the initial
fixation conditions are identical. Has anyone else encountered (and
hopefully solved) this problem?
Thanks in advance,
L. Charles Murtaugh
University of Utah
Dept. of Human Genetics
15 N. 2030 E. Rm. 4420B
Salt Lake City, UT 84113
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