title: Preservation of immunoreactivity and fine structure of adult C. elegans tissues using high-pressure freezing.
abstract: The location of a protein labeled by immunogold techniques can be resolved under an electron beam to within nanometers of its epitope, a resolution that makes immunoelectron microscopy a valuable tool for studies of cell biology. However, tissues in the nematode Caenorhabditis elegans are difficult to preserve for immunoelectron microscopic studies. The animal's cuticle slows the diffusion of solutions into the animal and thus makes it difficult to preserve both immunoreactivity and cell morphology. Here we describe a protocol that circumvents these problems. Specifically, we instantly immobilized tissue in vitreous ice by freezing living adult animals under high pressure. Frozen specimens were then chemically fixed, dehydrated, and embedded at low temperatures. As a result, chemical diffusion across the cuticle could occur over an extended period without morphological deterioration. We show that this method is capable of preserving both cell morphology, including fine structures, and immunoreactivity. Therefore, it provides a means to characterize the localization of endogenous proteins and exogenous proteins, such as the green fluorescent protein (GFP), with respect to subcellular compartments in C. elegans tissues by using postembedding immunogold labeling.
authors.affiliation: Biologie Cellulaire de la Synapse, Ecole Normale Supérieure, Paris, France.
authors.forename: Philippe
authors.initials: P
authors.lastname: Rostaing
authors.forename: Robby M
authors.initials: RM
authors.lastname: Weimer
authors.forename: Erik M
authors.initials: EM
authors.lastname: Jorgensen
authors.forename: Antoine
authors.initials: A
authors.lastname: Triller
authors.forename: Jean-Louis
authors.initials: JL
authors.lastname: Bessereau
mesh_terms: D000199:Actins / Q000648:ultrastructure; D000818:Animals; D017173:Caenorhabditis elegans / Q000378:metabolism / Q000648:ultrastructure*; D002451:Cell Compartmentation; D015925:Cryopreservation; D005629:Frozen Sections; D049452:Green Fluorescent Proteins; D007365:Intercellular Junctions / Q000648:ultrastructure; D008164:Luminescent Proteins / Q000378:metabolism; D016253:Microscopy, Immunoelectron / Q000379:methods; D008870:Microtubules / Q000648:ultrastructure; D009218:Myosins / Q000648:ultrastructure; D009474:Neurons / Q000378:metabolism / Q000648:ultrastructure; D015388:Organelles / Q000648:ultrastructure; D016707:Tissue Fixation
chemical_list: D000199:Actins; D008164:Luminescent Proteins; D049452:Green Fluorescent Proteins; D009218:Myosins