Biotinylated AMPA Receptor Labeling in Slices

Surface labelling of biotinylated AMPARs in slice tissue was performed as described in^{6 (link)}. 20 μΜ biotin (Sigma-Aldrich Cat# B4501) was added to the culture medium after SCE transfection. Four days later, slices were washed in HEPES-based aCSF (see SCE) and excess biotin was removed by dialysis in HEPES-based aCSF against 0.25 ml 2 mg/ml avidin Texas red (Thermo Fisher Scientific Cat# A820) through a Slide-A-Lyzer MINI (3500 molecular weight cut-off, Thermo Fisher) for 45 mins. Slices were then incubated for 45 min at room temperature in HEPES-based aCSF containing 120 nM Streptavidin-AF647 (Invitrogen). Slices were washed three times over 45 mins before fixation in phosphate-buffered saline (PBS) containing 4% paraformaldehyde and 4% sucrose (PFA, Sigma-Aldrich, Cat.# P6148) at 4 °C overnight. Slices were washed and stored in PBS until imaging. For imaging, slices were inverted on a glass-coverslip and imaged in PBS using a 63x/1.4NA oil-immersion objective on a commercial Leica TCS SP8 confocal microscope using LAS X software. Z-stacks of whole cells or whole stratum radiatum dendrites were taken, with EGFP, tdTomato and AF647 excited with 478 nm, 554 nm and 653 nm laser lines, with equivalent power settings across experimental conditions.