Immunofluorescence staining of phosphoinositides in primary mouse hippocampal neurons in dissociated culture

Phosphoinositides (PIPs) are low-abundant membrane lipids with critically important functions in cellular physiology. To investigate their subcellular distribution in neurons, we optimized protocols for immunofluorescence staining of intracellular or plasma membrane PIPs with commercially available antibodies. Here, we describe the preparation and transfection of primary mouse hippocampal neurons in dissociated culture, followed by immunofluorescence staining and quantitative analysis of PIP signals. In addition, we expand the application of the protocol to proteins located at the cytoplasmic leaflet of cellular membranes. For complete details on the use and execution of this protocol, please refer to Guo et al. (2022).