title: Immunoelectronmicroscopy of soluble and membrane proteins with a sensitive postembedding method.
abstract: The application of immunoelectronmicroscopy to soluble proteins is limited because soluble proteins can redistribute during fixation. Fixation may also adversely affect the recognition of proteins associated with membranes. We show here how displacements of soluble proteins can be prevented and antigen sensitivity improved by freeze-substitution immunocytochemistry. The usefulness of this method for soluble cytoplasmic proteins is demonstrated for the twitchin protein in Aplysia muscle and the kinesin motor proteins in squid giant axons, in which the sizes of various cytoplasmic pools of kinesins are estimated. The utility for membrane proteins present in small numbers of copies is demonstrated by labeling a glutamate receptor subunit in mouse cerebellar cortex and the ZO-1 protein in tight junctions between MDCK cells. Thus, freeze-substitution immunocytochemistry can show the native distribution of both soluble and membrane proteins labeled with polyclonal antibodies and, at the same time, can reveal structural features comparable to those in chemically fixed or osmium freeze-substituted samples.
authors.affiliation: Laboratory of Neurobiology, National Institute on Deafness and Other Communication Disorders, National Institutes of Health, Bethesda, Maryland, USA.
authors.forename: J E
authors.initials: JE
authors.lastname: Moreira
authors.forename: V
authors.initials: V
authors.lastname: Dodane
authors.forename: T S
authors.initials: TS
authors.lastname: Reese
mesh_terms: D000818:Animals; D001048:Aplysia; D001369:Axons / Q000737:chemistry / Q000648:ultrastructure; D029742:Caenorhabditis elegans Proteins; D002148:Calmodulin-Binding Proteins / Q000032:analysis; D002460:Cell Line; D002531:Cerebellum / Q000737:chemistry / Q000648:ultrastructure; D003598:Cytoskeletal Proteins / Q000032:analysis; D049832:Decapodiformes; D004285:Dogs; D017110:Freeze Substitution; D016591:Histocytological Preparation Techniques; D008565:Membrane Proteins / Q000032:analysis*; D051379:Mice; D008810:Mice, Inbred C57BL; D016253:Microscopy, Immunoelectron / Q000379:methods*; D009124:Muscle Proteins / Q000032:analysis; D009132:Muscles / Q000737:chemistry / Q000648:ultrastructure; D010750:Phosphoproteins / Q000032:analysis; D011506:Proteins / Q000032:analysis*; D017470:Receptors, Glutamate / Q000032:analysis; D012680:Sensitivity and Specificity; D016610:Tissue Embedding; D062826:Zonula Occludens-1 Protein
chemical_list: D029742:Caenorhabditis elegans Proteins; D002148:Calmodulin-Binding Proteins; D003598:Cytoskeletal Proteins; D008565:Membrane Proteins; D009124:Muscle Proteins; D010750:Phosphoproteins; D011506:Proteins; D017470:Receptors, Glutamate; C569043:Tjp1 protein, mouse; D062826:Zonula Occludens-1 Protein; C489233:unc-22 protein, C elegans