Immunofluorescent Staining Protocol for Cultured Cells

Cultured cells were fixed by immersion in 4% (w/v) PFA in PBS overnight at 4 °C and PBS washed before primary antibodies were applied in blocking solution (10% FCS/0.2% Triton X-100 in PBS) overnight at 4 °C. After PBS washing, Alexa Fluor-conjugated secondary antibodies, diluted in blocking solution, were applied overnight at 4 °C. Coverslips were washed thrice and mounted with DAKO fluorescent mounting medium (Sigma). Primary antibodies included: rabbit anti-Pcdh15^{32 (link)} [0.45 mg/ml; 1:200; Figs. 1 and 2], rabbit-anti-pan Pcdh15 (HL5614^{29 (link)}, 1:100; Fig. 8), rabbit anti-CD3 Pcdh15 (PB375^{29 (link)}, 1:100; Fig. 8), rat anti-GFP (Nacalai Tesque, 1:2000), goat-anti-PDGFRa (R&D System; 1:100), mouse-anti-Arp2 (Santa Cruz Biotechnology SC376698; 1:10) and mouse-anti-Arp3 (Santa Cruz Biotechnology SC48344; 1:10). Secondary antibodies included: donkey-anti-rat 488 (Invitrogen; 1:1000), donkey-anti-rabbit 568 (Invitrogen; 1:1000), donkey-anti-rabbit 647 (Invitrogen; 1:1000), donkey-anti-rabbit 488 (Invitrogen; 1:1000), donkey-anti-mouse 647 (Invitrogen; 1:1000) and donkey anti‐goat 647 (Invitrogen; 1:1000). Filamentous (F)-actin was also detected by exposing cells to Alexafluor-568 conjugated phalloidin (Invitrogen; 1:50) and cell nuclei were detected by exposing cells to Hoechst 33342 (Thermo Fisher Scientific; 1:1000).