Immunofluorescence Staining Protocols for Major Autophagy Proteins Including LC3, P62, and ULK1 in Mammalian Cells in Response to Normoxia and Hypoxia

Abstract

Immunofluorescence is an invaluable technique widely used in cell biology. This technique allows visualization of the subcellular distribution of different target proteins or organelles, by specific recognition of the antibody to the endogenous protein itself or to its antigen via the epitope. This technique can be used on tissue sections, cultured cells, or individual cells. Meanwhile, immunofluorescence can also be used in combination with non-antibody fluorescent staining, such as DAPI or fluorescent fusion proteins, e.g., GFP or YFP, etc.

Autophagy is a catabolic pathway in which dysfunctional organelles and cellular components are degraded via lysosomes. During this process, cytoplasmic LC3 translocates to autophagosomal membranes. Therefore, cells undergoing autophagy can be identified by visualizing fluorescently labeled LC3 or other autophagy markers. Immunofluorescence is an important part of autophagy detection methods even if observation of the formation of autophagosome by transmission electron microscopy has become a gold standard for characterizing autophagy.

By observing the immunofluorescence staining of some key autophagy proteins, we can intuitively evaluate the levels of autophagy in samples. Herein, this protocol describes the predominant method used for the research of autophagy, which mainly focuses on the immunofluorescence staining of cellular LC3, P62, and ULK1 in response to normoxia and hypoxia, by presenting the detailed materials required and methodology.