title: Correlative Microscopy for Localization of Proteins In Situ: Pre-embedding Immuno-Electron Microscopy Using FluoroNanogold, Gold Enhancement, and Low-Temperature Resin.
abstract: Immuno-electron microscopy (immuno-EM) is a technique that has been used widely to determine subcellular localization of proteins. Different approaches are available for immuno-EM: pre-embedding method, post-embedding, and cryosectioning (Tokuyasu "style"). Here we describe a pre-embedding technique that allows the labeling of a target protein in situ, retention of fluorescence signal in plastic, and its localization at the EM level in a given cellular context. The procedure can be technically challenging and labor intensive: it requires optimization of fixation protocols to better preserve the cellular morphology and screening of compatible antibodies. Nevertheless, immuno-EM can be a powerful localization tool.
authors.affiliation: National Center for Microscopy and Imaging Research, University of California San Diego, La Jolla, CA, 92093, USA, dboassa@ucsd.edu.
authors.forename: Daniela
authors.initials: D
authors.lastname: Boassa
mesh_terms: D000180:Acrylic Resins; D000818:Animals; D000906:Antibodies / Q000737:chemistry; D002460:Cell Line; D003080:Cold Temperature; D018031:Connexin 43 / Q000235:genetics* / Q000378:metabolism; D004847:Epithelial Cells / Q000378:metabolism* / Q000648:ultrastructure; D017629:Gap Junctions / Q000378:metabolism* / Q000648:ultrastructure; D015870:Gene Expression; D005976:Glutaral; D007668:Kidney / Q000166:cytology / Q000378:metabolism; D053768:Metal Nanoparticles / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods*; D016253:Microscopy, Immunoelectron / Q000379:methods*; D051381:Rats; D013266:Stilbamidines / Q000737:chemistry*; D016610:Tissue Embedding / Q000379:methods; D016707:Tissue Fixation
chemical_list: C049774:2-hydroxy-4,4'-diamidinostilbene, methanesulfonate salt; D000180:Acrylic Resins; D000906:Antibodies; D018031:Connexin 43; C580592:Gja1 protein, rat; D013266:Stilbamidines; C048707:LR white; D005976:Glutaral