Immunofluorescence Imaging of METTL7A Localization

Mock cells (either NMuMG or 4T1) or their stable transfectants overexpressing FLAG-tagged METTL7A1 were plated on glass chamber-slides (60 000 cells/well). Immunofluorescence was carried out 2 days after plating essentially as reported in (1 (link)). Rabbit polyclonal anti-METTL7A antibody (Abcam) and anti-FLAG antibody (F1804, Sigma) were used at a 1:500 dilution. Secondary antibodies, Alexa Fluor 488 anti-rabbit (Thermo Fisher) dilution 1:400, Alexa 488 anti-mouse (ThermoFisher) dilution 1:400, were diluted in IF buffer and incubated for 30 min at 37°C. ER Tracker dye for endoplasmic reticulum labeling was from Molecular Probes (E34251). Nuclei were counterstained with TO-PRO-3 (ThermoFisher). Images were collected using a three-channel TCS SP2 laser-scanning confocal microscope (Leica Microsystems). Images were imported into ImageJ to split and merge channels, cropped and adjusted for resolution and for intensity level range using Photoshop (scale bar = 10 μm).