A5750

Recording pipettes for cell attached recording contained 0.02% Lucifer yellow. After cell attached recordings were completed, negative pressure was applied to the recording pipette to rupture the membrane. Cells were held in the whole-cell configuration for 3–5 min to allow exchange of internal solution into the cell, and then a slight amount of positive pressure was applied to the pipette to detach it from the cell. Slices were then placed in 4% paraformaldehyde (PFA) in phosphate buffer (PB; 0.1 m sodium phosphate) and incubated overnight at 4°C. After fixation, slices were washed at room temperature in PB (3 × 5 min), and in Tris saline (TS) buffer (3 × 5 min) and blocked for 45 min in 10% normal horse serum (NHS)/0.3% Triton X-100/TS. After blocking, slices were incubated with primary antibody (rabbit anti-Lucifer yellow, 1:1000; Thermo Fisher Scientific A5750, RRID:AB_2536190) in 1% NHS/0.1% Triton X-100/TS for 18 h at 4°C. Slices were washed in TS (3 × 10 min) and then incubated with secondary antibody (donkey anti-rabbit Cy3, 1:500; Jackson ImmunoResearch 711-165-152, RRID: AB_2307443) in TS for 90 min at room temperature. Slices were washed in TS (3 × 10 min) and mounted on a charged microscope slide (Fisher 12-550-15) and dried completely before coverslipping with Prolong Gold antifade mounting media containing DAPI (ThermoFisher P36935).

Antibodies used in this study are as follows: Sema7A (goat, 1:3000, #AF-1835, R and D Systems); PlxnC1 (goat, 1:3000, discontinued, Abcam); CNG-A2 (rabbit, 1:200, #APC-045, Alomone Labs); vGlut2 (guinea pig, 1:1000, #AB2251-l, Millipore); GluR1 (rabbit, 1:1000, #ab51092, Abcam); GFP (rabbit, 1:1000, #A-10260, Thermo Fisher Scientific); Lucifer yellow (rabbit, 1:2000, #A-5750, Thermo Fisher Scientific); and EGR1 (rabbit, 1:1000, #ab6054, Abcam).