Labeling Postsynaptic Densities for Super-Resolution Microscopy With Minimal Signal-Loss and Offset.

Accurate labeling of excitatory postsynaptic sites remains a major challenge for high-resolution imaging due to the dense and sterically restricted environment of the postsynaptic density (PSD). Here, we present a protocol utilizing Sylites, 3 kDa synthetic peptide probes that bind with nanomolar affinity to key postsynaptic markers, PSD-95 and Gephyrin. eSylites (excitatory Sylites) specifically target the PDZ1 and PDZ2 domains of PSD-95, enabling precise and efficient labeling of excitatory postsynaptic density (ePSD). In contrast, iSylites (inhibitory Sylites) bind to the dimerizing E-domain of the Gephyrin C-terminus, allowing selective visualization of inhibitory postsynaptic density (iPSD). Their small size reduces linkage error and enhances accessibility compared to conventional antibodies, enabling clear separation of PSD-95 nanodomains in super-resolution microscopy. The protocol is compatible with co-labeling using standard antibodies and integrates seamlessly into multichannel immunocytochemistry workflows for primary neurons and brain tissue. This method enables robust, reproducible labeling of excitatory synapses with enhanced spatial resolution and can be readily adapted for expansion microscopy or live-cell applications. Key features • Protocol for using synthetic bidendate peptide probes: eSylites [1] for PSD-95 at excitatory synapses and iSylites [2] for Gephyrin at inhibitory synapses. • Compatible with standard antibody staining for multiplexed imaging of primary neurons and tissue sections. • Reduces linkage error, improving spatial resolution and nanodomain visualization in super-resolution microscopy.