Sc 28657

Retinal and scleral homogenates were resolved by SDS-PAGE and then blotted with specific antibodies for Ki67 (Abcam, ab15580, 1:200), TH (Millipore, AB152, 1:1000) and col1α1 (Santa Cruz, sc-28657, 1:1000) overnight at 4°C. Detection was achieved with an anti-rabbit-horseradish peroxidase (RD, HAF008, 1:1000). The signal was detected with a chemiluminescence kit (ECL, Thermo, 32106). Each protein band was normalized to β-actin expression levels in the same gel.

Cells were harvested by scraping in RIPA buffer (50 mM Tris-Cl, pH 7.4, 150 mM NaCl, 1 mM EDTA, 1% NP-40, 1 mM NaF, 1 mM Na₃VO₄, and 1 mM PMSF). Protein samples were separated by SDS-PAGE on 6%–15% acrylamide gels and transferred to PVDF membranes. The membranes were blocked with 5% skim milk in Tris-buffered saline containing 0.1% Triton X-100 for 1 h at room temperature. The membranes were incubated overnight at 4°C with specific primary antibodies and then hybridized with the secondary antibodies conjugated to horseradish peroxidase (HRP) for 1 h at room temperature. The reactions were detected by exposure to x-ray film after the application of the West-Q Chemiluminescent Substrate Plus Kit (GenDEPOT). The primary antibody dilutions were as follows: α-collagen I (1:2000, sc-28657, Santa Cruz), α-SMA (1:3000, A2547, Sigma-Aldrich), CTGF (1:1000, ab6992, Abcam), GLP-1R (1:250, ab39072, Abcam), and β-actin (1:5000, A5441, Sigma-Aldrich).

At 14 days post PDL cell seeding, the cells were fixed with 4% formaldehyde for 10 min, followed by permeabilized within PBS containing 0.2% Triton X-100 and blocked in PBS containing 1% bovine serum albumin (BSA, Sigma) for 1 h. Subsequently, cells incubated overnight at 4 °C either with a polyclonal rabbit to collagen type I antibody (sc-28657, Santa Cruz, CA, USA) or a polyclonal rabbit to osteocalcin antibody (sc-30044, Santa Cruz) at a dilution of 1:75 in PBS containing 1% BSA. After washing with PBS, cells were incubated for 1 h at 37 °C with TR-conjugated-goat-anti-rabbit antibodies (sc-2780, Santa Cruz) (1:100) diluted in PBS containing 1% BSA. Prior to viewing, samples were mounted with Vectashield containing DAPI nuclear staining (Vector, Burlingame, CA, USA). Images were captured from each surface with an OLYMPUS BX51 fluorescence microscope. The optical density (OD) of the fluorescent staining was quantified from 3 independent experiments using Image J software.