Fluorescence tracing of intracellular proteins.
Developments in fluorescence microscopy and the availability of fluorescently labeled antibodies and probes for localization of molecules and organelles have made the microscope an indispensable tool with which one can map specific molecules to subcellular loci allowing deep insight into cell and organelle biology. Furthermore, confocal microscopy permits analysis of the three dimensional architecture of cells that could not be accomplished by conventional light microscopy. The goal of fluorescence protein tracing by microscopy is to visualize cellular constituents and general cytoarchitecture as close to native organization as possible. To achieve this, and to preserve cellular structure in the best possible manner, the specimen is usually fixed chemically. Here I review several standard fixation, permeabilization and labeling schemes followed by examples of several standard imaging techniques.
Opas
M
M
Department of Anatomy & Cell Biology, University of Toronto, Ontario, Canada.
D000818:Animals; D002463:Cell Membrane Permeability; D005453:Fluorescence; D005456:Fluorescent Dyes; D006801:Humans; D007424:Intracellular Fluid / Q000737:chemistry; D008856:Microscopy, Fluorescence / Q000379:methods; D011506:Proteins / Q000032:analysis*; D016707:Tissue Fixation
D005456:Fluorescent Dyes; D011506:Proteins