[Histonet] h&e staining on frozen sections

[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
 
 
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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