Fluorescein isothiocyanate fitc

Isotype specific Zenon Mouse IgG labeling kits (Molecular Probes, Invitrogen, OR) were used to label primary antibodies. Following manufactures instructions, primary antibodies SWC9 (mature macrophage), CD16, SLA-DR class II DR, and CD163 (all from AbD Serotec, Raleigh, NC) were pre-labeled with Alexa Flour (A)594, A647, A405, and A430 (Table 1). After five minutes, non-specific IgG was added to block cross labeling. Mixtures were further diluted in PBS and then immediately used. Titration studies were performed using samples from whole blood and injured larynges to determine the appropriate concentrations of each label. The following conjugated anti-pig antibodies were also used simultaneously: CD14 or SWC3 (myeloid cells) labeled to fluorescein isothiocyanate (FITC; Abd Serotec) and human CD152 (CTLA–4) fused to murine IgG2a Fc labeled with RPE (Ancell Corporation, Stillwater, MN), which denotes expression of co-stimulatory markers CD80 and CD86. Isotype specific antibodies were used as controls, and each were labeled as described above.

Immunofluorescence for an endothelial cell marker (CD31) was performed on flattened retinal pigment epithelium (RPE) -choroid complexes 4 weeks after laser exposure to better assess CNV lesion size. Mice were sacrificed by CO₂ inhalation and eyes were harvested for tissue processing. Four radial incisions were made in the sclerochoroidal “cup” to prepare choroidal flat-mounts. Tissues were incubated in a 2%–4% paraformaldehyde solution (Panreac, Castellar del Vallés, Barcelona, Spain). The RPE-choroid-sclera complex was blocked with 5% donkey serum (DakoCytomation Denmark, Glostrup, Denmark) for 30 min and bathed in rat anti-mouse CD31 (1:50, DIA-310, Dianova, Hamburg, Germany) overnight at 4 °C. A donkey anti-rat antibody labelled with fluorescein isothiocyanate (FITC, 1:200, AbD Serotec, Puchheim, Germany) was then used to label CNV lesions. Tissue samples were observed with fluorescence microscopy (Axio Imager M1, Carl Zeiss, Germany), photographed, and analyzed by three independent, masked investigators. Image J software was used to measure the size of hyperfluorescent areas, which corresponded to CNV lesions.