[Histonet] h&e staining on frozen sections

From:"Charles Murtaugh"

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@genetics.utah.edu
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