Crude membrane proteins were isolated from 10 adult rat lenses or from 1 adult rat brain. Tissue was homogenized in 10 mL of 10 mM Tris-HCl (pH 7.4), 1 mM EDTA, 250 mM sucrose, 1 mM phenylmethylsulfonyl fluoride (PMSF), and 1 μg/mL leupeptin. The homogenate was centrifuged at 900g for 10 minutes at 4°C, and the resultant supernatant was centrifuged at 24,000 rpm (SW 40TI; Beckman Instruments, Fullerton, CA) for 75 minutes at 4°C. The pellet was solubilized by incubation for 1 hour in 500 μL buffer (100 mM Tris-HCl [pH 7.4], 1 mM EDTA, 0.5% TritonX-100, 0.5% sodium deoxycholate, and 1 mM PMSF). This homogenate was centrifuged at 14,000g for 10 minutes at 4°C, and the supernatant, containing the solubilized proteins, was collected in a separate tube. Proteins were separated on a 10% sodium dodecyl sulfate-polyacrylamide gel and transferred onto a nitrocellulose membrane by electrophoresis for 90 minutes at 170 mA. Transfer of proteins was confirmed by staining the nitrocellulose membrane for 4 minutes with Ponceau stain (1% Ponceau, 1% acetic acid in 100 mL filtered H2O (MilliQ; Millipore, Bedford, MA). Membranes were incubated overnight at room temperature in a blocking solution (1% BSA, 0.1% Tween20, 2 mM Tris-HCl, 140 mM NaCl [pH 7.6]) and subsequently incubated for 2 hours with goat anti-mdr protein antibody diluted 1:1000 (0.2 μg/mL in 1%BSA, Tris buffered saline [TBS]). Membranes were then exposed to biotinylated anti-goat IgG secondary antibody (Amersham) diluted 1:1000 for 1 hour, followed by streptavidin-horseradish peroxidase (HRP; Amersham) diluted 1:1000. After each incubation, the membranes were rinsed three times with water and washed three times for 15 minutes in TBS. The presence of P-glycoprotein was detected by enhanced chemiluminescence and exposed on autoradiographic film (Hyperfilm, ECL; Amersham).
For immunocytochemistry experiments, whole lenses were fixed for 16 hours in 2% paraformaldehyde in PBS. Wholemount capsules, with adherent epithelial cells, were fixed for 15 minutes. Lenses were briefly rinsed in PBS, incubated in 30% sucrose for 6 hours before incubating in optimal cutting temperature (OCT; Sakura Finetek, Tokyo, Japan) overnight. Whole lenses were mounted in OCT and frozen, and sections were cut at 16 μm. After three 5-minute washes in PBS, the lens sections or epithelial wholemounts were incubated in goat anti-mdr protein antibody diluted 1:50 (4 μg/mL in PBS) for 2 hours at room temperature. A control section, with goat anti-mdr protein antibody preincubated with peptide was also prepared. After three 5-minute washes in PBS, the sections were incubated with a secondary antibody, anti-goat IgG conjugated to red dye (Rhodamine Red; Molecular Probes, Eugene, OR) diluted 1:100. Lens sections were washed with three 15-minute washes in 1× PBS before the sections were mounted onto slides with antifading reagent (FluoroGuard; Bio-Rad, Richmond, CA). Sections were viewed with a confocal laser scanning microscope (TCS 4D, Leica Lasertechnik, Heidelberg, Germany) fitted with an argon-krypton mixed gas laser. The software accompanying the microscope was used to record confocal microscopy images (SCAware; Leica).