Rabbit polyclonal anti lc3b

Paraffin-embedded tissue sections were incubated in the oven at 52 C overnight. On the following day, tissue sections were deparaffinized and rehydrated with graded ethanol dilutions. Antigen retrieval in sodium citrate buffer (10 mM, pH 6.0) was followed by three washing steps. After washing the slides with PBS, tissues were blocked with blocking solution at room temperature for 1 hour. Immunofluorescence staining was performed by incubating the paraffin sections with polyclonal rabbit anti-LC3B (1:50; Cell Signaling Technology), monoclonal rabbit anti-ATG5 (1:50; Gene-Tex, Irvine, CA), polyclonal rabbit anti-p62 (1:100; Sigma-Aldrich), polyclonal rabbit anti-ATG7 (1:50; Abgent, San Diego, CA), monoclonal rabbit anti-cathepsin B (1:500; Cell Signaling Technology), monoclonal rabbit anti-cathepsin D (1:150; Cell Signaling Technology), and monoclonal mouse anti-cathepsin L (1:200; Abcam) in blocking buffer. After 24 hours, tissue sections were washed in PBS and incubated with secondary antibody (goat anti-rabbit 532; Molecular Probes, Invitrogen, Carlsbad, CA) at room temperature for 1 hour. Subsequently, slides were washed with PBS. Images were acquired by confocal laser scanning microscopy (LSM 510 or LSM 800; Carl Zeiss AG, Jena, Germany), and the area of the epidermis was analyzed by ImageJ software or Imaris (Bitplane AG, Zurich, Switzerland).