Alexa fluor 633 secondary antibody

ReN VM cells were cultured as described above. Cells were fixed in 4% formaldehyde for 15 minutes and permeabilized with 0.1% Triton X-100 for 20 minutes. Permeabilized cells were incubated overnight at 4°C in PBS buffer containing 1% BSA and primary antibody directed against ATP1A1 (catalog number ab7671, Abcam, Waltham, MA; used at 1:200). Following three PBS washes, Alexa-Fluor 633 secondary antibody (catalog number A-21052, Invitrogen, Burlington, ON; used at 1:400) was incubated with cells for 90 minutes at ambient temperature in PBS buffer containing 1% BSA. Cells were washed three times in PBS and mounted onto glass slides using ProLong Gold containing DAPI (catalog number P36934, Invitrogen, Burlington, ON). Z-stack images were captured using an LSM880 inverted confocal microscope (Carl Zeiss Canada Ltd, Toronto, ON) and processed using Zen Blue software (Carl Zeiss Canada Ltd).

Mouse monoclonal anti-hemagglutinin (HA) antibody was from BioLegend (Clone 16B12, Cat. No. 901513). Polyclonal GFP antibody was from Novus Biologicals (Cat. No. NB600-308). Rabbit polyclonal anti-phospho cofilin antibody was from Cell Signaling Technology (Cat. No. C02-3311S). Mouse monoclonal anti-cofilin antibody was from Santa Cruz Biotechnology (Cat. No. sc-376476). Mouse monoclonal anti-actin antibody was a gift from Dr José Manuel Hernández (CINVESTAV-IPN). Alexa-Fluor 633 secondary antibody was from Invitrogen (Cat. No. A21052). 4′,6-diamidino-2-phenylindole (DAPI) and Phalloidin-Rhodamine reagents were from Thermo Fisher Scientific (Cat. No. D1306 and R415, respectively). Mouse monoclonals anti-α-tubulin and anti-c-MYC antibodies were from Developmental Studies Hybridoma Bank (clone 12G10 and 9E10, respectively). Antibodies were used at a 1:1000 ratio unless otherwise specified. Unless otherwise stated, chemical reagents were from Merck-Sigma-Aldrich.