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Summary: 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 : Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.