Re: [Histonet] h&e staining on frozen sections

From:"Bryan Hewlett"


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.


Bryan Hewlett

----- Original Message ----- 
From: "Charles Murtaugh" 
Sent: Saturday, April 30, 2005 12:03 AM
Subject: [Histonet] h&e staining on frozen sections

Hi all--

  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,

  Charles Murtaugh


L. Charles Murtaugh

University of Utah
Dept. of Human Genetics
15 N. 2030 E. Rm. 4420B
Salt Lake City, UT 84113

tel 801-581-5958
fax 801-581-7796
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