Goat anti mouse af488

HEK293T cells were grown in 6-well plates with 22 mm square no. 1.5 coverslips coated with poly-D-lysine hydrobromide (Sigma-Aldrich, #P6407). Cells in each well were transiently transfected using 100 ng V2R and 900 ng sheared salmon sperm DNA (Invitrogen) and 1:3 25 kDa linear Polyethylenimine (Polysciences, #23966). The cells were grown for 20-28h. The cells were fixed using 3% paraformaldehyde, incubated for 20 min, and washed 2x with PBS. Coverslips were then elevated from 6-well plates and cells permeabilised with 0.5% IGEPAL in PBS for 5 min at 4ºC, washed 2x 5 min with PBS, then blocked with 3% BSA-1% glycine in PBS, incubated for 30 min, washed 2x 5 min with PBS, then blocked with 10% Goat serum (Abcam, #ab7481), incubated 30 min, then added polyclonal anti-SNAP antibody (Thermo-Fisher,# CAB4255) 1:100 and Anti-P4HB antibody [RL90] (abcam, #ab7481) 1:500 in 10% Goat serum and incubated overnight at 4 C. Washed coverslip 3x in PBS and incubated cover slips in goat-anti-rabbit-AF-647 (Abcam, #ab150083) 1:500, goatanti-mouse-AF-488 (Abcam, #ab150117) 1:500, DAPI stain 5 mg/mL 1:1000 in 10% Goat serum in the dark for 1h. Washed coverslips 3x5min with PBS and mounted coverslips with Vectashield antifade mounting medium (Vectorlabs, #H-1000).