Mouse anti cftr

For immunostaining, CFPAC6.0 cells were seeded onto a cover-glass placed inside a well of a multiwell plate. The coverglass was washed twice with 1 × phosphate-buffered saline and then fixed for 25 min in 4% paraformaldehyde and 20mM formic acid solution (Sigma-Aldrich, St Louis, MO, USA) made in Grace’s media (Lonza, Allendale, NJ, USA). Cells were washed three times for 10 min with antibody wash buffer (1 × phosphate-buffered saline: 0.1% Triton X-100: 0.2% bovine serum albumin) and incubated overnight at 4 °C in primary antibody (mouse anti-CFTR, 1:500, R&D Systems). They were then washed three times for 10 min and incubated for 2 h at room temperature in secondary antibody (anti-mouse IgG_2A Alexa 488, Molecular Probes, Invitrogen, Carlsbad, CA, USA). After washing two times for 10 min in antibody wash, rhodamine phalloidin (Molecular Probes, Invitrogen) was added for 15 min. After washing two times for 5 min, 4’,6-diamidino-2-phenylindole was added for 10 min, then removed and then the coverglass was mounted with Vectashield (Vector Laboratories Inc., Burlingame, CA, USA). Samples were imaged using a Zeiss 710 confocal microscope and 63× Plan Apo numerical aperture 1.4 lens. The image depicts Z-stacks, and an average of 300 cells in 10 different fields was analyzed.