Sc 20082

Whole mount retinae were fixed in 4% PBS-buffered formalin for 3 hours at room temperature. After washing, retinae were blocked and permeabilized in 1% BSA and 0.5% Triton X-100 overnight at 4°C. Following three washes with PBS buffer (pH 6.8) containing 0.5% Triton X-100, 1 mmol/L CaCl₂ and 1 mmol/L MnCl₂, the retinae were incubated with biotin conjugated isolectin B4 (1:100, Sigma-Aldrich, L1240, Taufkirchen/Munich, Germany) overnight. Primary antibodies against CD11b (mouse anti-rat, AbD SeroTec, MCA275G, 1:100 dilution) and CD74 (rabbit anti-rat, Santa Cruz, SC-20082, 1:100 dilution) were diluted in blocking buffer as above and incubated at 4°C overnight. The secondary antibodies porcine anti-rabbit TRITC (DAKO, R0156, 1:20 dilution), goat anti-mouse Alexa Fluor^® 488 (Invitrogen, A21131, 1:200 dilution), and streptavidin Alexa Fluor^® 633 (Invitrogen, S21375, 1:500 dilution) were incubated at room temperature for one hour. All images were scanned using Leica TCS SP2 confocal microscope. Microglia were defined as expressing CD11b⁺ and CD74⁺ and quantified in ten randomly selected fields (400x magnification) from superficial and deep vascular retinal layers.

Cells grown on coverslips were fixed with 4% paraformaldehyde in PBS for 15 min at room temperature and permeabilized with 0.1% Triton X-100 in PBS for 20 min at room temperature. For immunostaining, the cells were incubated with various primary antibodies at 37 °C for 1 h, washed three times with PBS and incubated with appropriate secondary antibody, anti-rabbit IgG conjugated with Dylight405 (711-475-152, 1:1,000, Jackson ImmunoResearch, West Grove, PA) or anti-mouse conjugated with Alexa488 (715-545-150, 1:1,000, Jackson ImmunoResearch) at 37 °C for 1 h. The coverslips were washed three times with PBS and incubated with anti HLA-DP monoclonal antibody conjugated with DyLight594 (H1584, 1:200, Leinco Technologies) at 37 °C for 1 h, washed three times with PBS and mounted on a slide with antifade medium (Vectashield, Vector Labs, Burlingame, CA). Fluorescence images were captured using a confocal microscope (Zeiss LSM700). The primary antibodies utilized were as follows: rabbit anti-Ii polyclonal antibody (sc-20082, 1:200, Santa Cruz Biotechnology), mouse anti-Ii monoclonal antibody (sc-6262, 1:200, Santa Cruz Biotechnology), mouse anti-EEA1 monoclonal antibody (610457, 1:250, BD Biosciences), mouse anti-LAMP1 monoclonal antibody (sc-20011, 1:100, Santa Cruz Biotechnology) and mouse anti-PDI monoclonal antibody (MA3-019, 1:500, Thermo Scientific).