Flow cytometry analysis of GST tag on HEK-293 cells transiently expressing GST-GAD65. Cells were fixed with 70% ethanol for 10 minutes, permeabilized with 0.25% Triton™ X-100 for 20 minutes, and blocked with 5% BSA for 1 hour at room temperature. Cells were labeled with GST tag mouse monoclonal antibody (Cat. No. 136700, red histogram) or with mouse isotype control (pink histogram) at 1-3 µg/million cells in 2.5% BSA. After incubation at room temperature for 2–3 hours, the cells were labeled with Alexa Fluor 488-conjugated rabbit anti-mouse secondary antibody (Cat. No. A11059) at a dilution of 1:400 for 30 minutes at room temperature. The representative 10,000 cells were acquired and analyzed for each sample using an Applied Biosystems Attune NxT Flow Cytometer. The purple histogram represents unstained control cells and the green histogram represents no-primary-antibody control.