Rapid embedding methods into epoxy and LR White resins for morphological and immunological analysis of cryofixed biological specimens. 2014

Kent L McDonald
Electron Microscope Laboratory, University of California, Berkeley, CA 94720, USA.

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.

UI MeSH Term Description Entries
D007150 Immunohistochemistry Histochemical localization of immunoreactive substances using labeled antibodies as reagents. Immunocytochemistry,Immunogold Techniques,Immunogold-Silver Techniques,Immunohistocytochemistry,Immunolabeling Techniques,Immunogold Technics,Immunogold-Silver Technics,Immunolabeling Technics,Immunogold Silver Technics,Immunogold Silver Techniques,Immunogold Technic,Immunogold Technique,Immunogold-Silver Technic,Immunogold-Silver Technique,Immunolabeling Technic,Immunolabeling Technique,Technic, Immunogold,Technic, Immunogold-Silver,Technic, Immunolabeling,Technics, Immunogold,Technics, Immunogold-Silver,Technics, Immunolabeling,Technique, Immunogold,Technique, Immunogold-Silver,Technique, Immunolabeling,Techniques, Immunogold,Techniques, Immunogold-Silver,Techniques, Immunolabeling
D004853 Epoxy Resins Polymeric resins derived from OXIRANES and characterized by strength and thermosetting properties. Epoxy resins are often used as dental materials. Epoxy Resin,Resin, Epoxy,Resins, Epoxy
D000818 Animals Unicellular or multicellular, heterotrophic organisms, that have sensation and the power of voluntary movement. Under the older five kingdom paradigm, Animalia was one of the kingdoms. Under the modern three domain model, Animalia represents one of the many groups in the domain EUKARYOTA. Animal,Metazoa,Animalia
D001419 Bacteria One of the three domains of life (the others being Eukarya and ARCHAEA), also called Eubacteria. They are unicellular prokaryotic microorganisms which generally possess rigid cell walls, multiply by cell division, and exhibit three principal forms: round or coccal, rodlike or bacillary, and spiral or spirochetal. Bacteria can be classified by their response to OXYGEN: aerobic, anaerobic, or facultatively anaerobic; by the mode by which they obtain their energy: chemotrophy (via chemical reaction) or PHOTOTROPHY (via light reaction); for chemotrophs by their source of chemical energy: CHEMOLITHOTROPHY (from inorganic compounds) or chemoorganotrophy (from organic compounds); and by their source for CARBON; NITROGEN; etc.; HETEROTROPHY (from organic sources) or AUTOTROPHY (from CARBON DIOXIDE). They can also be classified by whether or not they stain (based on the structure of their CELL WALLS) with CRYSTAL VIOLET dye: gram-negative or gram-positive. Eubacteria
D016610 Tissue Embedding The technique of placing cells or tissue in a supporting medium so that thin sections can be cut using a microtome. The medium can be paraffin wax (PARAFFIN EMBEDDING) or plastics (PLASTIC EMBEDDING) such as epoxy resins. Embedding, Tissue
D017110 Freeze Substitution A modification of the freeze-drying method in which the ice within the frozen tissue is replaced by alcohol or other solvent at a very low temperature. Substitution, Freeze
D018515 Plant Leaves Expanded structures, usually green, of vascular plants, characteristically consisting of a bladelike expansion attached to a stem, and functioning as the principal organ of photosynthesis and transpiration. (American Heritage Dictionary, 2d ed) Plant Leaf,Leaf, Plant,Leave, Plant,Leaves, Plant,Plant Leave

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