Protocol for performing 3D-STORM-based nanoscale organization of NMDA receptors in hippocampal brain tissue.

Direct stochastic optical reconstruction microscopy (dSTORM) unveils ionotropic N-methyl-D-aspartate receptor (NMDAR) organization into discrete nanometer-size domains (nanoclusters) within the postsynaptic density (PSD) of glutamatergic synapses, tuning receptor signaling. Here, we present a protocol to perform 3D-dSTORM imaging of the NMDAR in organotypic and acute rat hippocampal brain slices by combining conventional dSTORM with fluorescent self-interference. We describe steps for sample preparation, immunohistochemistry, 3D-dSTORM acquisition, and image analysis to successfully super-resolve NMDAR nanodomains in three dimensions. For complete details on the use and execution of this protocol, please refer to Kellermayer et al.,1 Bon et al.,2 and Ferreira et al.3.