title: Rapid embedding methods into epoxy and LR White resins for morphological and immunological analysis of cryofixed biological specimens.
abstract: A variety of specimens including bacteria, ciliates, choanoflagellates (Salpingoeca rosetta), zebrafish (Danio rerio) embryos, nematode worms (Caenorhabditis elegans), and leaves of white clover (Trifolium repens) plants were high pressure frozen, freeze-substituted, infiltrated with either Epon, Epon-Araldite, or LR White resins, and polymerized. Total processing time from freezing to blocks ready to section was about 6 h. For epoxy embedding the specimens were freeze-substituted in 1% osmium tetroxide plus 0.1% uranyl acetate in acetone. For embedding in LR White the freeze-substitution medium was 0.2% uranyl acetate in acetone. Rapid infiltration was achieved by centrifugation through increasing concentrations of resin followed by polymerization at 100°C for 1.5-2 h. The preservation of ultrastructure was comparable to standard freeze substitution and resin embedding methods that take days to complete. On-section immunolabeling results for actin and tubulin molecules were positive with very low background labeling. The LR White methods offer a safer, quicker, and less-expensive alternative to Lowicryl embedding of specimens processed for on-section immunolabeling without traditional aldehyde fixatives.
authors.affiliation: Electron Microscope Laboratory, University of California, Berkeley, CA 94720, USA.
authors.forename: Kent L
authors.initials: KL
authors.lastname: McDonald
mesh_terms: D000818:Animals; D001419:Bacteria; D004853:Epoxy Resins; D017110:Freeze Substitution / Q000379:methods*; D007150:Immunohistochemistry / Q000379:methods*; D018515:Plant Leaves; D016610:Tissue Embedding / Q000379:methods*
chemical_list: D004853:Epoxy Resins