After a 2-hour treatment period with H
2O
2, RNA isolation from cultured RPE cells was performed after an interval of 48 hours to allow RNA generation with respect to published data.
26 The concentration of hydrogen peroxide was reduced to 150 μM to diminish lethal oxidative stress to RPE cells. Total RNA was isolated by the guanidium thiocyanate-phenol-chloroform extraction method (Stratagene, Heidelberg, Germany). Structural integrity of the total RNA samples was confirmed by electrophoresis on 1% agarose gels. Total RNA (2 μg) was denatured and size fractionated by gel electrophoresis in 1% agarose gels containing 2.2 M formaldehyde. The RNA was then vacuum-blotted onto a nylon membrane (Roche, Indianapolis, IN) and cross-linked (1600 μJ, Stratalinker; Stratagene). To assess the amount and quality of RNA, the membrane was stained with methylene blue, and images were obtained (LAS-1000; RayTest, Pforzheim, Germany). Prehybridization was performed at 68°C for 1 hour. Hybridizations were performed at 68°C overnight in prehybridization solution (Dig Easy Hyb; Roche) containing 50 ng/mL digoxygenin-labeled, caveolin-1–specific sense 5′-GAGCTGAGCGAGAAGCAAGT-3′ and antisense 5′-ACAGCAAGCGGTAAAACCAG-3′ riboprobe. Riboprobes were synthesized as previously described.
27 After hybridization, the membrane was washed twice with 2 × SSC, 0.1% sodium dodecyl sulfate (SDS) at room temperature (RT), followed by two washes in 0.1 × SSC, 0.1% SDS, for 15 minutes at 68°C. After hybridization and posthybridization washes, the membrane was washed for 5 minutes in washing buffer (100 mM maleic acid [pH 7.5], and 150 mM NaCl, 0.3% Tween-20) and incubated for 60 minutes in blocking solution. The blocking solution contained 100 mM maleic acid (pH 7.5), 150 mM NaCl, and 1% blocking reagent (Roche). Anti-digoxigenin alkaline phosphatase (Roche) was diluted 1:10,000 in blocking solution, and the membrane was incubated for 30 minutes. After four additional washes in washing buffer (15 minutes each), the membrane was equilibrated in detection buffer (100 mM Tris-HCl, 100 mM NaCl [pH 9.5]) for 5 minutes. For fluorescence detection, a chemiluminescence substrate (CDP-Star; Roche) was diluted 1:100 with detection buffer, and the filter was incubated for 5 minutes at RT. After air drying, the semidry membrane was sealed in a plastic bag. Chemiluminescence was detected with the imager (LAS-1000; RayTest). Exposure times ranged between 5 and 40 minutes. Quantification of the chemiluminescence signal was performed on computer (AIDA software; RayTest).