15 mm round cover glass slide

Cells with 90% confluent were seeded into a six-well culture plate. After gene transfection or siRNA interference was performed, the cells were cultured for another 24 h. Then the cells with a concentration of 5 × 10⁴ cell/ml (500 μl/well of the cell suspension) were seeded into a 24-well culture plate. Each bottom of the 24 wells were pre-inserted a 15 mm round cover glass slide (NEST Biotechnology Co., China) and the cells were cultured for 24 h to form a confluent monolayer. The cells were washed with ice-cold PBS and fixed with 4% formaldehyde (Solarbio, Beijing, China) for about 20 min. After the permeability treatment with PBS containing 0.1% Triton X-100 (Solarbio, Beijing, China) for 15 min and blocked with 3% bovine serum albumin for 1 h at 37°C. Subsequently, the coverslips were incubated with CAB39 (1:50; Cell Signaling, USA) or GLUT1 (1:50; Proteintech) antibody at 4°C overnight. After incubation with CoraLite488-conjugated Affinipure Goat Anti-Rabbit IgG (H+L) secondary antibody (SA00013-2, PTG, Beijing, China) for 1 h, the cover slips were mounted in Prolong Gold antifade reagent with DAPI (Beyotime, Shanghai, China) for 5 min, and coverslipped. Images were obtained on a confocal microscope (Carl Zeiss, Germany)