Phosphoinositide detection at synapses of fixed murine hippocampal neurons

The minor phospholipid phosphatidylinositol 4,5-bisphosphate [PI(4,5)P₂] is crucial for neurotransmission and has been implicated in Parkinson's disease. Here, we present a staining protocol for the analysis of activity-dependent changes of PI(4,5)P₂ at synapses. We describe steps for stimulating and fixing murine hippocampal neurons, staining with probes for PI(4,5)P₂ and a synaptic marker, and analysis by high-resolution microscopy. Our approach gives insights into local PI(4,5)P₂ synthesis and turnover at synapses and can be extended to phosphoinositide lipids other than PI(4,5)P₂. For complete details on the use and execution of this protocol, please refer to Bolz et al.¹.