Dmi 8 inverted epi uorescence microscope

Cells were quickly washed with ice-cold PBS and xed in 4% paraformaldehyde (PFA, Electron Microscopy Sciences, Hat eld, PA, USA) and blocked for 30 minutes at room temperature (RT) in presence of PBS supplemented with 10% goat serum (ThermoFisher) supplemented with 0.2% Triton-X100 (Sigma). Cells were incubated overnight at 4ºC in primary antibodies targeting BCRP (1:100, Millipore, RRID: AB_11213795), claudin-5 (1:100, Life Technologies, RRID: AB_2533200), GLUT1 (1:100, ThermoFisher, AB_10979643), GLUT3 (1:100, ThermoFisher, AB_2809974), GLUT4 (1:100, ThermoFisher, AB_11153908), MRP1 (1:100, Millipore, RRID: AB_2143819), occludin (1:100, Life Technologies, AB_2533101) and P-gp (1:50, ThermoFisher, AB_1233253) diluted in 10% goat serum (PBSG). Primary antibodies detection occurred by incubation with goat-anti mouse Alexa Fluor® 555-conjugated secondary goat anti-mouse (Life Technologies) for 1 hour at room temperature. Cells were observed at 200X magni cation (20X longdistance dry objective) and acquired using a Leica DMi-8 inverted epi uorescence microscope (Leica Microsystems, Wetzlar, Germany). Images were processed using ImageJ (Image J, NIH, Bethesda, MD).
Relative uorescence was quanti ed using the built-in function in ImageJ. Background uorescence was subtracted from unlabeled cells incubated with the secondary antibody only.

Cells were quickly washed with ice-cold PBS and xed with 100% cold methanol or 4% paraformaldehyde, followed by blocking in PBSG (PBS with 10% normal goat serum (ThermoFisher)) with 0.2% Triton-X100 (Sigma-Aldrich) for 1h at room temperature. Cells were incubated in presence of primary antibodies targeting GLUT-1 (SPM498 clone, RRID:AB_10979643, 1:100; ThermoFisher), GLUT-3 (RRID:AB_10694437, 1:100;; ThermoFisher), GLUT4 (RRID:AB_11153908, 1:100; ThermoFisher), occludin (RRID: AB_2533101, 1:100, ThermoFisher), and claudin-5 (AB_2533200, 1:100, ThermoFisher) overnight at 4°C, followed by detection using Alexa-Fluor-conjugated secondary antibodies (ThermoFisher) for 1h at room temperature. Cells were observed using Leica DMI8 inverted epi uorescence microscope (Leica Microsystems, Buffalo Grove, IL, USA). Micrograph images were processed and analyzed using ImageJ (NIH, Bethesda, MD, USA).