Immunostaining of Primary Hippocampal Neurons

Immunostaining of rat primary hippocampal neurons or HT-22 cells employed established protocols (Baumert et al., 2020 (link)). Briefly, cells cultured on poly-D-lysine-coated coverslips were fixed with 4% paraformaldehyde for 10 min at room temperature and then permeabilized with 0.5% Triton X-100 solution for 15 min. Following fixation and permeabilization, cells were blocked with PBS containing 1% bovine serum albumin overnight at 4°C. Cells were then incubated with the indicated primary antibodies overnight at 4°C and subsequently rinsed three times with fresh PBS for 10 min each, followed by an overnight rinse with PBS at 4°C. Cells were then incubated with Alexa Fluor fluorescent dye-conjugated secondary antibodies (Invitrogen) for 1 h at room temperature before the coverslips were washed and finally mounted onto glass slides with Vectashield (Vector Laboratories) mounting solution. Cells were visualized using a Nikon T2i Inverted Confocal Microscope with an Apo-Plan 60 × 1.4 NA oil objective at room temperature. Images were captured with a Nikon A1-DUG GaAsP hybrid four-channel multi-detector and Nikon NIS-Elements software. All z-series images were acquired at a pixel size of 100 nm and a step size of 0.2 μm.