Rabbit polyclonal anti cox 2

Eyes were harvested from mice and fixed in 4% paraformaldehyde for 5 hours at room temperature. For cryopreservation, fixed eye samples were placed in sucrose gradients 10%, 15%, and 20% for 1 hour each at room temperature. Eye samples were placed in an OCT compound and frozen at −80°C. The 10-μm thick eye sections were cut using a cryostat. Sections were blocked with 10% normal donkey serum with 0.5% Tween-20 for 1 hour. Sections were stained with primary antibodies (rabbit polyclonal anti-iNOS [Santa Cruz Biotechnology, CA, USA], rabbit polyclonal anti-COX2 [Abcam, Cambridge, MA, USA], and rabbit polyclonal immunoglobulin G (IgG) control [Jackson Immunoresearch, West Grove, PA, USA]) at 1:650 dilution at 4°C overnight. To detect CD45⁺ cells, rat antimouse CD45 (1:500 dilution; BD Biosciences) was added to the sections at 4°C overnight. Sections were stained with secondary antibodies, fluorescein isothiocyanate goat antirabbit (Jackson Immunoresearch) at 1:100 dilution and DI594 goat antirat IgG (Jackson Immunoresearch) at 1:10000 dilution for 90 minutes. Hoechst deoxyribonucleic acid dye (Thermo, Rockford, IL, USA) at 1:1000 was used to stain nuclei. Retinas were imaged using a fluorescence microscope.