title: Immunoelectron Microscopy of Cryofixed Freeze-Substituted Yeast Saccharomyces cerevisiae.
abstract: Immunolabeling electron microscopy is a challenging technique with demands for perfect ultrastructural and antigen preservation. High-pressure freezing offers an excellent way to fix cellular structure. However, its use for immunolabeling has remained limited because of the low frequency of labeling due to loss of protein antigenicity or accessibility. Here we present a protocol for immunogold labeling of the yeast Saccharomyces cerevisiae that gives specific and multiple labeling while keeping the finest structural details. We use the protocol to reveal the organization of individual nuclear pore complex proteins and the position of transport factors in the yeast Saccharomyces cerevisiae in relation to actual transport events.
authors: Department of Biology of the Cell Nucleus, Institute of Molecular Genetics AS CR, v.v.i., Videnska 1083, 142 20, Prague 4, Czech Republic.
authors: Jindřiška
authors: J
authors: Fišerová
authors: School of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, UK.
authors: Christine
authors: C
authors: Richardson
authors: School of Biological and Biomedical Sciences, Durham University, South Road, Durham, DH1 3LE, UK. m.w.goldberg@durham.ac.uk.
authors: Martin W
authors: MW
authors: Goldberg
mesh_terms: D000906:Antibodies / Q000737:chemistry; D015925:Cryopreservation / Q000379:methods*; D004853:Epoxy Resins / Q000737:chemistry; D005404:Fixatives / Q000737:chemistry; D017110:Freeze Substitution / Q000379:methods*; D015870:Gene Expression; D005976:Glutaral / Q000737:chemistry; D007150:Immunohistochemistry / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods; D008867:Microtomy; D028861:Nuclear Pore Complex Proteins / Q000235:genetics / Q000378:metabolism; D012441:Saccharomyces cerevisiae / Q000378:metabolism / Q000648:ultrastructure*; D029701:Saccharomyces cerevisiae Proteins / Q000235:genetics / Q000378:metabolism; D013194:Staining and Labeling / Q000379:methods; D016610:Tissue Embedding / Q000379:methods*; D016707:Tissue Fixation / Q000379:methods
chemical_list: D000906:Antibodies; D004853:Epoxy Resins; D005404:Fixatives; D028861:Nuclear Pore Complex Proteins; D029701:Saccharomyces cerevisiae Proteins; D005976:Glutaral
keywords: Freeze substitution; Immunostaining; Lowicryl; Nuclear; Transmission electron microscopy; Transport; Yeast