Mct4 sc 50329

Cells grown on Transwell^® inserts were initially fixed with methanol:acetone for 10 min at room temperature, washed with PBS followed by apical membrane biotinylation with biotin anti-goat IgG (1:200 dilution) and labelled with streptavidin conjugated to Alexa Fluor 594 (red) or 488 (green) (1:200 dilution). The apical surface was washed with PBS and cells blocked and permeabilized with 1% bovine serum albumin containing 0.1% Triton in Tris-buffered saline. Primary antibodies for MCT1 (sc-365501) and MCT4 (sc-50329), were from Santa Cruz, USA. MCT2 (ab198272) and MCT3 (ab60333), were from AbCam, UK. Cells were incubated with 1:100 dilution of antiserum and incubated at room temperature for 1 hour. Cells were washed with PBS and the secondary antibody was added (anti-mouse Texas Red or anti-rabbit Alexa Fluor 488 at 1:100 dilution) for 30 min at room temperature. After rinsing with PBS, Transwell^® inserts were removed with a scalpel and mounted onto slides with Vectashield mounting medium containing 4′,6-diamino-2-phenylindole (DAPI) for nuclei staining. Images were visualised using a Zeiss LSM 510 Meta confocal microscope.