Ab 450 na

Choroidal lysates were prepared from samples of the C57BL/6 mice taken at the indicated time interval after laser injury. Protein samples, quantified using NanoDrop 2000 UV-Vis Spectrophotometer (30 µg/each lane; Thermo Fisher Scientific, Inc.) were dissolved in Laemmli buffer (cat. no. 1610737; Bio-Rad Laboratories, Inc., Hercules, CA, USA), boiled for 3–4 min, and centrifuged at 4°C for 2 min at 20,000 × g to remove insoluble materials. A total of 30 µg protein per lane was separated by SDS-PAGE (12%) and transferred to 0.2 µm nitrocellulose membranes. The blocked membranes were probed overnight (4°C) with goat anti-VEGF (1:200; sc-152-G; Santa Cruz Biotechnology, Inc.), goat anti-HIF-1α (1:200; sc-12542Y-15; Santa Cruz Biotechnology, Inc.), goat anti-SDF-1α (1:200; sc-7427; Santa Cruz Biotechnology, Inc.), goat anti-CCL3 (1:1,000; AB-450-NA; R&D Systems) or goat anti-GAPDH antibodies (1:200; sc-48166; Santa Cruz Biotechnology). The membranes were then incubated at RT for 1 h with a horseradish peroxidase-conjugated rabbit anti-goat secondary antibody (1:5,000; 5160–2504; R&D Systems), and immunoreactive bands were visualized using ECL reagent (Advansta, Menlo Park, CA, USA). The intensities of the protein bands were quantified and normalized to GAPDH using Image J Software version 2.1.4.7 (National Institutes of Health, Bethesda, MD, USA).