[Histonet] h&e staining on frozen sections
Charles Murtaugh
murtaugh <@t> genetics.utah.edu
Fri Apr 29 23:03:18 CDT 2005
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 dreadful.
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
email murtaugh <@t> genetics.utah.edu
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