A method for preparing quick-frozen, freeze-substituted cells for transmission electron microscopy and immunocytochemistry.
A quick-freeze, freeze-substitution method is described which employs glutaraldehyde as well as osmium tetroxide (OsO4) in a 'double-fixation' protocol comparable to that used for conventional transmission electron microscopy. Cultured cells are quick-frozen in Freon 22 and freeze-substituted in an ethanolic solution of glutaraldehyde. Specimens destined for TEM are postfixed in OsO4 in acetone, embedded in Epon-Araldite, and sectioned. This method yielded ultrastructural preservation which was comparable to that obtained from methods employing OsO4 alone as a freeze-substitution fixative. However, if glutaraldehyde is used alone as a freeze-substitution fixative, specimens can be processed for immunocytochemistry without additional treatment with permeabilizing agents.
D000199:Actins / Q000032:analysis; D000818:Animals; D002478:Cells, Cultured; D002642:Chick Embryo; D005404:Fixatives; D005455:Fluorescent Antibody Technique; D005629:Frozen Sections; D005727:Ganglia, Spinal / Q000648:ultrastructure*; D005976:Glutaral; D008854:Microscopy, Electron / Q000379:methods*; D008858:Microscopy, Phase-Contrast; D008867:Microtomy; D009457:Neuroglia / Q000648:ultrastructure; D009474:Neurons / Q000648:ultrastructure; D009993:Osmium Tetroxide; D014335:Tropomyosin / Q000032:analysis
D000199:Actins; D005404:Fixatives; D014335:Tropomyosin; D009993:Osmium Tetroxide; D005976:Glutaral