2.7. Immunofluorescence

Cells were grown on glass coverslips at a density of 2 × 10⁴ cells/well and incubated at 37 °C and 5% CO₂ overnight. Then, cells were incubated in DMEM without FBS for 24 h. Immunofluorescence was performed as previously described [26]. Briefly, slides were incubated with the primary antibodies (room temperature, overnight), and then incubated with the secondary antibody anti-rabbit-Alexa Fluor 488 (A11008, Invitrogen, Waltham, MA, USA, 1:500) for 1 h in 5% BSA (MCT4 and MCT1), or the secondary antibody anti-rabbit-Alexa Fluor 594 (A11032, Invitrogen USA, 1:250) (HKII and HIF-1α). Images were acquired by a fluorescence microscope (Olympus IX81) with the Cell P software.