Bullfrog sacculi were fixed in 3% paraformaldehyde in cold PBS for 25 min, washed in PBS, and permeabilized in 0.1% TX-100 in PBS for 1 h. Following a PBS wash, the tissue was blocked in 5% goat serum in PBS for 1 h and incubated overnight at 4°C in 4.5 μg/ml HCS-1 antibody in blocking solution. Bound antibodies were detected with Alexa-488 goat anti-mouse secondary antibody (Invitrogen), and filamentous actin was labeled with 33 nM Alexa-568 phalloidin (Invitrogen). Samples were mounted in Vectashield (Vector Laboratories, Burlingame, CA, USA) and viewed with a LSM 510 META confocal microscope (Zeiss). Isolated bullfrog saccular hair cells (Lumpkin and Hudspeth 1995 (link)) were allowed to settle onto coverslip dishes (Mattek, Ashland, MA, USA) coated with 1 mg/ml concanavalin A. The cells were fixed for 25 min in 3% paraformaldehyde in cold PBS, washed for 10 min in PBS, and permeabilized for 20 min in 0.1% saponin in PBS. Dishes were washed for 5 min in PBS and blocked for 1 h in 10 mg/ml bovine serum albumin (fraction V; EMD Biosciences, San Diego, CA, USA) and 5% goat serum in PBS. Primary antibody solutions of 4.5 μg/ml HCS-1 antibody with or without 2.5 μg/ml F2b anti-plasma membrane Ca2+ ATPase b isoform (anti-PMCAb) antibody (Dumont et al. 2001 (link); gift from P. Gillespie, Oregon Health & Science University) in blocking solution were incubated with the cells overnight at room temperature. Following two 10-min washes with PBS/0.1% Tween-20, dishes were incubated at room temperature for 2 h with blocking solution containing 33 nM Alexa-568 phalloidin and either 13 μg/ml Alexa 488 goat anti-mouse antibody alone to detect bound mAb HCS-1 or 13 μg/ml goat anti-mouse Alexa 488 and 13 μg/ml Alexa-568 goat anti-rabbit (Invitrogen) to visualize both otoferlin and PMCAb. Dishes were then washed 2 × 10 min in PBS/0.1% Tween-20 and 1 × 10 min in PBS and mounted in Vectashield. Images were obtained using an LSM 510 META confocal microscope with a 100×, 1.4 NA objective.