Immunofluorescence Microscopy of the Mammalian Golgi Apparatus

Immunofluorescence is a technique that uses antibodies and fluorophores to label structures inside cells. The cells are normally fixed and permeabilized, and then structures are labelled using primary antibodies directly conjugated to fluorophores, or, more commonly, first with an antibody against an antigen of interest followed by a secondary antibody conjugated to a fluorophore that binds to the primary antibody. Fluorescence can be visualized using widefield, confocal, or super-resolution microscopy. Here we focus on labelling of the Golgi apparatus and show that different fixation and permeabilization conditions can significantly affect labelling of Golgi proteins and describe how to optimize fluorescent detection of Golgi proteins.