Anti lc3ii

All reagents were from Sigma unless noted. Saccharomyces cerevisiae (14 (link)) was kindly provided by Dr. van Haan (Vu University, Amsterdam, The Netherlands). β-glucan particles (GP) were produced from zymosan particles as previously described (15 (link)). These particles specifically activate Dectin-1 and do not engage Toll-like receptors. Other reagents include anti-LC3II (immunoblotting, MBL International clone 115; immunofluorescence, MBL International clone 153), anti-phospho Syk (immunoblotting and immunofluorescence, Cell Signaling), anti-phospho p40phox (Cell Signaling), anti-p40phox (Millipore), anti-GAPDH (Santa Cruz), anti-LAMP1 (Santa Cruz), and anti-GFP (Life Technology).

Enucleated eyes were fixed in 4% paraformaldehyde at room temperature for 3 h, the anterior segment, and the posterior portion of the eye post-fixed in the same fixative overnight at 4°C before being placed in 25% sucrose for a second overnight period at 4°C for cryoprotection. Eyecups were embedded in OCT compound (Sakura Finetec, Torrance, CA) and cut into 10-μm thick sections. Primary antibodies (1:50) were: anti-LC3-II (MBL International, Japan), anti-LAMP (Abcam, Cambridge, MA), and biotin-conjugated mouse monoclonal anti-Thy-1 (BD-Pharmingen, San Jose, CA). Sections were exposed to the appropriate secondary antibodies: goat anti-mouse IgG FITC-conjugate (1:500; Southern Biotechnology, Birmingham, AL), or goat anti-rabbit IgG fluorescein-conjugate (1:500; Invitrogen, Carlsbad, CA). Anti-fade mounting media containing DAPI (EMC Biosciences, La Jolla, CA) was applied and sections cover-slipped.