Af2065

Whole eyes were fixed in 10% phosphate buffered formalin, paraffin embedded and sectioned. PASH and GMS staining were performed by the CWRU Visual Science Research Center histology core. For immunohistochemistry, sections were treated with proteinase K (Dako 2015-011, Carpinteria, CA) and blocked in 1.5% serum. Mouse S100A9 polyclonal Ab (2 µg/ml, R&D Systems AF2065, Minneapolis, MN), Mouse S100A8 polyclonal Ab (2 µg/ml R&D Systems AF3059), or Anti-mouse neutrophil antibody NIMP-R14 (20 µg/ml) were used, followed by staining with Alexafluor 488 Chicken anti-goat IgG (1:2000, Life Technologies A2467, Grand Island, NY) or Alexafluor 488 Goat anti-rat IgG (1:250, Life Technologies A11006). Slides were imaged at 200–400X. Neutrophil quantification from histological sections was obtained by measuring % NIMP-R14 positive area/cornea using Metamorph software.

Corneas were dissected at 24–48h pi and treated with 1X collagenase for 1–2 h. Cell suspensions were washed in 1X PBS + 5% FBS, incubated with anti-mouse CD16/CD32 (clone 93, eBioscience 16–0161-86, San Diego, CA)(to block Fc receptors) and stained with anti-mouse neutrophil antibody NIMP-R14-PE (abcam ab125259, Cambridge, MA) or isotype for 1h and live/dead fixable far read stain (Thermo Fisher). For intracellular staining, cells were fixed and permeabilized using an intracellular staining kit (eBioscience). Cells were incubated with Mouse S100A9 polyclonal Ab ( R&D Systems AF2065), Mouse S100A8 polyclonal Ab ( R&D Systems AF3059), or isotype for 45 min, washed and incubated with Alexafluor 488 Chicken anti-goat IgG (1:2000, Life Technologies A2467) for 30 min, washed, resuspended in FACS buffer and analyzed in a BD Accuri C6 (San Jose, CA). Analysis was performed using Accuri C6 software. Cells were gated on FSC/SSC, followed by live cells.