Ab115574

RPE and neuroretina samples were extracted with a RIPA buffer (1% Triton X-100, 100 mmol/l Tris-HCl, pH 7.5; 150 mmol/l NaCl; 1.5 mmol/l EDTA, pH 8.0; 25 mmol/l NaF; 0.5 mmol/l Na₃VO₄; 0.1 mmol/l phenylmethylsulfonyl fluoride; and Complete protease inhibitors [Roche, Basel, Switzerland]) and homogenized by sonication. Protein concentrations were determined using bicinchoninic acid assay (Bio-Rad Laboratories, Madrid, Spain). Equivalent amounts (20 µg) of total protein extracts were resolved by 10% SDS-PAGE and transferred to ECL nitrocellulose membranes (Hybond, Amersham Pharmacia Biotech). The membranes were incubated with a primary antibody against VAP-1 (1:1000; ab115574, Abcam, Madrid, Spain) and further incubated with a peroxidase-conjugated secondary antibody (Bio-Rad Laboratories, Madrid, Spain). Membranes were stripped and re-probed with β-actin to evaluate the lane-loading control. Proteins were visualized using the enhanced chemiluminescence detection system ECL (Advansta, CA).

Paraffinized eyes were cut at a thickness of 7 μm. Sections were deparaffinized with xylene, rehydrated in ethanol, washed in PBS, and placed in an antigen-retrieval solution (Dako A/S, Glostrup, Denmark). Permanganate bleaching was used to eliminate the autofluorescence of the RPE. Sections were then incubated for 1 h with 2% BSA in 0.05% Tween in PBS to block unspecific binding and then incubated overnight at 4 °C with the anti-VAP-1 antibody (1:500; ab115574, Abcam, Madrid, Spain) or anti-collagen IV (1:200; ab6586, Abcam, Madrid, Spain). After washing, sections were incubated with Alexa Fluor 488 and 594 secondary antibodies (Molecular Probes, Invitrogen, Madrid, Spain) at room temperature for 1 h. Slides were coverslipped with a mounting medium containing 4′, 6-diamidino-2-phenylindole (DAPI) for the visualization of cell nuclei (Vector Laboratories, Palex, Sant Cugat del Vallés, Spain). Images were acquired with a confocal scanning microscope (FV1000, Olympus, Hamburg, Germany).