Lc3b rabbit polyclonal

T80 and HEY cells were seeded onto glass coverslips (250,000 cells per well), allowed to adhere overnight, treated with 250μM FAC, and processed according to previously published methodology ^{[6 (link)]}. For colocalization studies, cells were fixed in 4% formaldehyde (in PBS), followed by blocking in 5% goat serum and 0.1% Triton-X-100 (in PBS) for 1h at room temperature. The cells were then stained with anti-TFEB mouse monoclonal (#H00007942, 20μg/mL, Abnova (Taipei City, Taiwan)) antibody overnight in a humidified chamber at 4°C followed by a 1h incubation with AlexaFluor-488 anti-mouse antibody (#A12379, 1:500, Fisher Scientific (Pittsburgh, PA, USA)). Next, the fixed cells were incubated with either anti-LAMP1 (#9091, 1:200, Cell Signaling Technology (Danvers, MA, USA)) or LC3B rabbit polyclonal (#2775, 1:2000, Cell Signaling Technology (Danvers, MA, USA)) antibody overnight in a humidified chamber at 4°C followed by 1h incubation with AlexaFluor-546 anti-rabbit secondary (#A11035, 1:500, Fisher Scientific (Pittsburgh, PA, USA)). Slides were viewed and imaged at 63X magnification using a PerkinElmer UltraVIEW Confocal Spinning Disc Microscope (PerkinElmer Incorporation). Three independent experiments were performed. Representative images were captured and colocalization was quantified via Pearson Correlation Coefficient using the Velocity Software (Version 6.1.1).