X-Message-Number: 10850
Date: Sat, 28 Nov 1998 12:19:59 -0800 (PST)
From: Doug Skrecky <>
Subject: modern mummification of rat brain

Authors
  van Lookeren Campagne M.  Oestreicher AB.  van der Krift TP.  Gispen WH. 
  Verkleij AJ.
Institution
  Department of Pharmacology, Rudolf Magnus Institute, University of Utrecht,
  The Netherlands.
Title
  Freeze-substitution and Lowicryl HM20 embedding of fixed rat
  brain: suitability for immunogold ultrastructural
  localization of neural antigens.
Source
  Journal of Histochemistry & Cytochemistry.  39(9):1267-79, 1991 Sep.
Abstract
  We examined the suitability of freeze-substitution and Lowicryl HM20
  embedding of aldehyde-fixed rat brain to localize several
  neural antigens at the ultrastructural level. The following rabbit polyclonal
  and mouse monoclonal antibodies were used: affinity-purified polyclonal
  immunoglobulins G raised to B-50/GAP43 (a membrane-anchored,
  growth-associated protein); affinity-purified polyclonal immunoglobulins G to
  human glial fibrillary acidic protein (GFAP; a subunit of glial filaments); a
  polyclonal antiserum raised to adrenocorticotropic hormone[25-39] (a
  neuropeptide present in dense-core granules); a polyclonal antiserum raised
  to myelin basic protein (a protein present in compact myelin of the central
  nervous system); and mouse monoclonal antibodies to synaptophysin (an
  integral membrane protein of small synaptic vesicles). Rat mesencephalon was
  fixed by perfusion with buffered 2% glutaraldehyde and 4% paraformaldehyde,
  cryoprotected, and frozen in liquid nitrogen. Freeze-substitution of tissue
  was performed with anhydrous methanol and 0.5% uranyl acetate at -90 degrees
  C. Semi-thin Lowicryl sections were used for light microscopic visualization
  of B-50 in the ventromedial mesencephalic central gray substance. The
  procedure preserves well the ultrastructure of this region and the
  immunoreactivity of the selected antigens. This study shows that dehydration
  by freeze-substitution, combined with Lowicryl HM20 embedding at sub-zero
  temperature, provides a successful method of preparation of fixed
  brain tissue for ultrastructural studies, allowing
  immunogold localization of several neural antigens by double labeling in the
  same section and in serial sections.

Rate This Message: http://www.cryonet.org/cgi-bin/rate.cgi?msg=10850