title: Freeze-substitution and Lowicryl HM20 embedding of fixed rat brain: suitability for immunogold ultrastructural localization of neural antigens.
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.
authors.affiliation: Department of Pharmacology, Rudolf Magnus Institute, University of Utrecht, The Netherlands.
authors.forename: M
authors.initials: M
authors.lastname: van Lookeren Campagne
authors.forename: A B
authors.initials: AB
authors.lastname: Oestreicher
authors.forename: T P
authors.initials: TP
authors.lastname: van der Krift
authors.forename: W H
authors.initials: WH
authors.lastname: Gispen
authors.forename: A J
authors.initials: AJ
authors.lastname: Verkleij
mesh_terms: D000818:Animals; D001921:Brain / Q000648:ultrastructure; D001923:Brain Chemistry; D002148:Calmodulin-Binding Proteins / Q000032:analysis; D015925:Cryopreservation; D019922:GAP-43 Protein; D007150:Immunohistochemistry; D008297:Male; D008562:Membrane Glycoproteins / Q000032:analysis*; D009419:Nerve Tissue Proteins / Q000032:analysis*; D051381:Rats; D011919:Rats, Inbred Strains; D016610:Tissue Embedding / Q000379:methods*
chemical_list: D002148:Calmodulin-Binding Proteins; D019922:GAP-43 Protein; D008562:Membrane Glycoproteins; D009419:Nerve Tissue Proteins