Rabbit anti lc3a

Primary human fibroblasts (IMR90 cells) were grown on glass cover slips, treated as appropriate, and fixed with 4% paraformaldehyde. Unspecific epitopes were blocked with 3% BSA before permeabilization with 0.1% Triton X-100 in PBS. The fixed cells were then incubated overnight with primary antibodies diluted in PBS containing 1% BSA: rabbit anti-LC3A (1:200, from Cell Signaling, Danvers, MA, USA; order number 4599), mouse anti-LC3B (1:200, from NanoTools, Teningen, Germany; order number 0260-100), rabbit anti-LC3C (1:200, from Cell Signaling, Danvers, MA, USA; order number 14736), guinea pig anti-p62 (1:400, from Progen, Heidelberg, Germany; order number GP62-C), rabbit anti-LAMP2 (1:1000, from BioCat, Heidelberg, Germany; order number WA-ABV10697.100). The cells were subsequently incubated with Cy2-, Cy3- and Cy5-coupled secondary antibodies (1:500, from Jackson Immunoresearch, West Grove, PA, USA) for 2 h at room temperature. Cell nuclei were counterstained with 1 µg/mL 4,6-diamidino-2-phenylindole (DAPI, from Sigma-Aldrich, St. Louis, MO, USA). The cells were then analyzed and photographed using a confocal laser-scanning microscope (TCS SP5 from Leica Microsystems, Wetzlar, Germany). All pictures were taken with a 63x objective and 2x zoom at the highest possible resolution (2048 × 2048 pixels).