For immunohistochemistry assays, enucleated eyes were fixed using 4% paraformaldehyde in 0.1 M phosphate buffer pH 7.4 (PB), for 1 hour at room temperature. To maintain retinal orientation, a suture was placed on the superior pole of each eye before enucleation. Following several washes with PB, the eyes sequentially were cryoprotected in 15% and 20% (wt/vol) sucrose for 1 hour, and 30% sucrose overnight. Next day, the cornea, lens, and vitreous were dissected out, and the eyecups were embedded in Tissue-Tek (Sakura Finetek, Torrance, CA, USA), and frozen in liquid N2. Then, 16-μm vertical retinal cryosections along the nasotemporal axis were mounted on slides (Superfrost Plus; Menzel GmbH & Co. KG, Braunschweig, Germany), washed, and incubated with blocking solution (5% normal donkey serum in PB containing 0.5% Triton X-100) for 1 hour at room temperature. Immunostaining with a cocktail of mouse anti-MHC class-II RT1B monoclonal antibody, 1:50 (clone OX-6; AbD Serotec, Kidlington, United Kingdom), and rabbit anti-Iba1 polyclonal antibody, 1:500 (Wako Chemicals, Neuss, Germany) was performed overnight at room temperature in a wet chamber. After incubation with the primary antibody, retinal sections were washed and incubated in a mix of secondary antibodies: Alexa Fluor 555-conjugated donkey anti-mouse IgG (Molecular Probes, Eugene, OR, USA) and Alexa Fluor 488-conjugated donkey anti-rabbit IgG, both at a 1:100 dilution, for 1 hour. Finally, the sections were washed and mounted with Citifluor (Citifluor Ltd., London, UK) for visualization under a laser scanning confocal microscope (TCS SP2; Leica Microsystems, Wetzlar, Germany). Immunohistochemical controls were performed by omission of either the primary or the secondary antibodies. Only retinal sections that contained the optic nerve were used for cell scoring.