temperature. Sections were washed again and mounted in mounting medium and coverslipped. Macrophages were also detected by F4/80 antibody in the choroid-RPE flat mounts on Day 3. All the procedures were performed at 4uC. In brief, 3 mice in each group were sacrificed and the eyes were enucleated and fixed with 4% paraformalde- hyde for 30 minutes. Then washed three times in PBS, followed by dehydration and rehydration through a methanol series (40%, 80%, 100%, 80%, 40%). Washing again, eye cups were obtained by removing the anterior segments. After blocking with 5% BSA for 1 hour, eye cups were incubated 48 hours with F4/80 antibody. After five washings with PBS (1 hour each time), the eyecups were incubated with Alexa 546–tagged secondary antibodies and DAPI overnight. The eyecups were washed again and flat-mounted as previously mentioned. These slides were examined under a fluorescence microscope (AX80; Olympus, Tokyo, Japan). Flow cytometry of macrophages In order to quantify the number of macrophages, eyes were enucleated on Day 3. The RPE-choroid complexes were separated and disrupted with a sharp micro-scissor into small pieces. Then they were treated with collagenase D (2 mg/mL, Roche, Germany) on a Bio-shaker at 37uC for 1 hour. After that, they were filtered, and the single-cell suspensions were incubated in Fc block (1:100, Catalog No. 14-0161, eBiosciences, USA) for 15 minutes on ice, then stained with FITC-conjugated anti-mouse F4/80 (1:30, Catalog No. 11-4801, eBiosciences, USA) or FITC- conjugated isotype (1:100, Catalog No. 11-4321, eBiosciences, USA). Live cells were detected by gating on forward versus side scatter [4], followed by analysis of F4/80 in the fluorescence channel (FACSCalibur; BD Biosciences, USA). At least 50,000 viable cells were analyzed per condition. Data were analyzed by the system software (Cellquest software; BD Biosciences, USA). A total of 5 mice were examined per group. The number of ocular- infiltrating macrophages was calculated from the percent of each population in the gate of the precounted, total number of viable cells using trypan blue dye exclusion. Macrophage sorting and quantitative real-time PCR analysis (qPCR) of VEGF For macrophage sorting, eyes were enucleated on Day 3. Single cells were isolated and stained as the described above. Cells showing FITC-F4/80 signals were collected by a FACSAria flow cytometer (BD Biosciences, USA) with FlowJo software (Tree Star. OR, USA). Total RNA was extracted from sorted macrophages in each group using the RNeasy Plus Mini kit (Catalog No. 74134 Qiagen, Valencia, CA) and transcribed into cDNA using ExScript RT reagent kit (Takara Bio, Otsu, Shiga, Japan) according to the manufacturer’s protocol. Real-time PCR analysis was performed by Platinum SYBR Green qPCR SuperMix-UDG (Catalog No. 11733-038, Invitrogen, Carlsbad, CA). The reaction was carried out for 40 cycles of 15 seconds at 95uC and 1 minute at 60uC after an initial incubation at 95uC for 10 minutes. The levels of PCR products were monitored with the Mx3000P QPCR System (Stratagene, USA). The baseline and threshold were adjusted according to the manufacturer’s instructions. The relative abundance of transcripts was normalized using either the expression level of GAPDH mRNA or VEGF-A mRNA by DDCt method. Three individual gene-specific values thus calculated were averaged. The primers used in this experiment are as follows- Mouse VEGF-A: sense 59-AGCCGAGCTCATGGACGGGT-39 and antisense 59-AGTAGCTTCGCTGGTAGACATC-39; Mouse CCR2 Blocker Inhibits Choroidal Neovascularization GAPDH: sense 59-TGGCAAAGTGGAGATTGTTGCC-39 and antisense 59-AAGATGGTGATGGGCTTCCCG-39. At least 8 eyes were needed to obtain a sufficient number of enriched macrophages for qPCR analysis in the above process. Enzyme-linked immunosorbent assay (ELISA) of VEGF To quantify VEGF protein levels, the RPE-choroid complexes were micro-surgically isolated from the eyes on Day 3, and placed immediately into 100 ml RIPA buffer (R0278, Sigma) supple- mented with 1% Protease inhibitor cocktail (P8340, Sigma) at 4uC. After mechanical disruption, lysates were placed on ice for 15 minutes, and centrifuged at 14,000 rpm for 10 minutes at 4uC. The supernatants were collected and preserved at 270uC. Protein concentrations were determined by Coomassie Bradford Protein Assay Kit (Catalog No. 23200, Pierce, USA). The VEGF levels in the supernatant were determined by mouse VEGF ELISA kit (Quantikine; R&D Systems) at 450 nm to 570 nm, with an absorption spectrophotometer (ARVOTM MX 1420 multilabel counter, PerkinElmer, Kanagawa, Japan), and normalized to total protein, according to the manufacturer’s protocols. Two eyes were needed to extract one protein sample, and eight mice in each group were examined. Western blot analysis of ERK phosphorylation To determine whether INCB3344 treatment affected the MAPK signaling pathway in the laser-induced CNV model, the activation of ERK1/2 was assayed by ERK1/2 phosphorylation in the choroid-RPE complex using western blot analysis on Day 3. The protein extraction and the calculation of protein concentration were the same as the ELISA protocols. Eight mg of the total protein per sample was diluted with Laemmli Sample Buffer (Catalog No. 161- 0737, Bio-Rad, CA), heated at 95uC for 5 min, separated by SDS- PAGE (Multigel?Mini, Cosmo Bio, Tokyo, JP), and electroblotted onto polyvinylidene fluoride membrane (PVDF, GE Healthcare, Buckinghamshire, UK). After blocking with 2.5% skim milk for 1 hour at room temperature, the membranes were incubated with a rabbit polyclonal anti-phospho-ERK antibody (1:2000, Catalog No. 4370, Cell Signaling, Danvers, Massachusetts), a rabbit polyclonal anti-ERK to detect total ERK protein (1:1000, Catalog No. 4695, Cell Signaling), or anti-GAPDH (14C10) (1:1000, Catalog No. 2118, Cell Signaling) over night at 4uC. After washing with 0.1% Tris-buffered saline (TBS)-Tween, blots were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:2500, Catalog No. 7074, Cell Signaling) for 1 h at room temperature. The blots were then washed three times with 0.1% TBS-Tween and the signals were visualized by an ECL kit (GE Healthcare, Buckinghamshire, UK) according to the manufactur- er’s protocol. The densities of immunoreactive bands were measured using Image J for Windows (NIH, Bethesda, Maryland). Eight mice in each group were examined. Effects on established CNV We also evaluated the effect of INCB3344 on already established CNV as well as laser-induced, newly formed CNV. Spontaneous CNV was detected in 10–14 months old Sod12/2 mice by fundus photography and fluorescence angiography (FA) using a digital camera (CCD Color Video Camera, Sony, Japan) and recorded by IMAGEnet 2000 Digital Image System (Topcon, Japan). The fluorescein sodium solution (10%; 0.1 ml/kg; Fluorescite; Alcon, Fort Worth, TX) was injected into the intraperitoneal cavity of the mice. CNV was defined as present when early hyperfluorescence with late leakage was present at the site of dyed lesion during fundus examination. To evaluate drug treatment, 6 Sod12/2 mice with CNV received intravitreal PLoS ONE | www.plosone.org 3 December 2011 | Volume 6 | Issue 12 | e28933