IF analyses of the RPE-choroid-sclera flat mounts were performed as previously described (16). Enucleated eyes were fixed with 4% paraformaldehyde for 30 min at room temperature (RT). For IF staining of sectioned retina and choroid, fixed eyeballs were then dehydrated in 20% sucrose solution overnight, embedded in tissue-freezing medium (Leica), and cut into 20-μm sections. Samples were blocked with 5% donkey or goat serum in PBST (0.3% Triton X-100 in phosphate-buffered saline) and then incubated in blocking solution with one or more of the following antibodies at 4°C overnight: rabbit anti-human CD31 polyclonal antibody (#AB32457, abcam), goat anti-human Tie2 polyclonal antibody (#AF313, R&D systems), hamster anti-mouse CD31 monoclonal antibody (#MAB1398Z, clone 2H8, Millipore), goat anti-mouse Tie2 polyclonal antibody (#AF762, R&D systems), chicken anti-GFP polyclonal antibody (#AB16901, Millipore), rat anti-mouse CD144 monoclonal antibody (#740933, clone 11D4.1, BD Biosciences), rabbit anti-mouse opsin polyclonal antibody (#AB5405, Millipore), and rabbit anti-mouse rhodopsin monoclonal antibody (#27182, clone D4B9B, Cell Signaling Technology). After several washes, the samples were incubated for 4 hours at RT with the following secondary antibodies (Jackson ImmunoResearch Laboratories): fluorescein isothiocyanate (FITC)– or Cy5-conjugated anti-hamster immunoglobulin G (IgG), FITC- or Cy3-conjugated anti-rabbit IgG, FITC- or Cy3-conjugated anti-goat IgG, FITC-conjugated anti-chicken IgG, and Cy3-conjugated anti-human IgG. Samples were mounted with fluorescent mounting medium (VectaShield H-1000; Vector Laboratories), and IF images were acquired with a confocal microscope (LSM880; Carl Zeiss Microscopy). CD31+ CNV volumes were measured using the MATLAB image processing toolbox (MATLAB 8.1; MathWorks), which are presented in cubic micrometers. The volume of CNV in one eye was averaged to give one experimental value. Morphometric analyses of choriocapillaris were performed using ImageJ software (NIH). The relative area of choriocapillaris was calculated as a percentage of the CD31+ area divided by its control area. To quantify the expression of Tie2 and CD144, intensities were measured in the CD31+ choriocapillary area. To perform IF staining for hypoxia, mice were intraperitoneally injected with pimonidazole hydrochloride (60 mg/kg) in PBS (Hypoxyprobe Green Kit; Hypoxyprobe) and sacrificed after 1 hour. An FITC-conjugated monoclonal antibody against pimonidazole was used for IF staining of the flat-mounted RPE-choroid-sclera complex. For statistical analysis, values from four random spots at the center of the choroid in each eye were averaged. For comparison of staining intensities, the values were normalized by the background signals in nonvascularized regions, and their ratios were normalized by control and presented as percentages.

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