X-Message-Number: 12439
Date: Sun, 19 Sep 1999 10:49:14 -0700 (PDT)
From: Doug Skrecky <>
Subject: freezing and fixation of rat cerebral cortex

Authors
  Reger JF.  Escaig F.
Institution
  Department of Anatomy and Neurobiology, University of Tennessee, Memphis.
Title
  A comparative fine
  structure study of rat cerebral cortex following ultra-rapid
  freezing and conventional chemical fixation procedures.
Source
  Journal of Submicroscopic Cytology & Pathology.  20(4):691-700, 1988 Oct.
Abstract
  Adult rat (Sprague-Dawley) cerebral cortex was processed by ultra-rapid
  freezing at liquid helium temperature followed by freeze-substitution, osmium
  fixation and by other chemical, post-osmication procedures at 0-4 degrees and
  ambient temperatures as a comparative study for purposes of
  identifying differences and/or similarities in fine
  structure following these techniques. Five methods of
  processing were used: 1) rapid, slam-freezing at liquid helium temperature
  followed by osmium tetroxide/acetone freeze substitution; 2) perfusion with
  buffered, 2% glutaraldehyde at ambient temperature followed by
  post-osmication (2%); 3) en-bloc, buffered 2% glutaraldehyde fixation at 0-5
  degrees centigrade and post-osmication (2%); 4) buffered, 2% osmium tetroxide
  perfusion at ambient temperature; and 5) en-bloc, buffered 2% osmium
  tetroxide fixation at 0-5 degrees centigrade. In ultra-rapid-frozen cortex
  good preservation was seen to a depth of 10-15 microns from the surface of
  the initial, copper-block contact. The tissue processed by
  ultra-rapid-freeze, freeze substitution demonstrates a general 'smoothness'
  of plasmalemmal and organelle membranes not observed in tissue prepared by
  chemical fixation alone. Cellular and organelle morphological differences
  were minor beyond the general 'smoothness' of membranes and a more intense
  background, electron density found in tissue prepared by rapid-freeze. Of
  particular interest was the practically identical images found in the four,
  chemical techniques not preceded by ultra-rapid freezing. High magnification
  images also revealed rather minor differences following ultra-rapid-freezing
  compared to tissue fixed by chemical fixation alone. Although these
  morphological differences are minimal, there can be no question of the fact
  that ultra-rapid-freeze followed by freeze-substitution is morphologically
  superior to chemical fixation alone. Ultra-rapid-freeze, eventually utilizing
  other substitution agents than osmium tetroxide, will offer several
  advantages and should be particularly useful for investigators involved in
  cytochemical and immunochemical methods.

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