Orca 2 ccd camera

ARPE-19 cells were grown on coverslips, fixed and permeabilized with methanol at −20 °C for 10 min, and then non-specific sites were blocked by incubation with 3% BSA in Tris-buffered saline and Triton X-100 (TBST) (20 mM Tris, pH 7.5, 150 mM NaCl, 2 mM EGTA, 0.1% Triton X-100) for 30 min. The cells were incubated with anti-acetylated-tubulin antibody to detect primary cilia (1:1000; T6793; Sigma-Aldrich; St. Louis, MO). Acetylated tubulin is a well-accepted marker for primary cilia. Cells were then incubated with FITC-conjugated donkey anti-mouse secondary antibody (1:200; 715–095-150; Jackson ImmunoResearch Inc.; West Grove, PA). DNA was stained with 4′,6-diamidino-2-phenylindole (DAPI) incorporated into the mounting media (Vector Labs; Burlingame, CA). The intracellular localization of proteins was observed with a Nikon E600 fluorescence microscope, Pan Fluor 100× objective (N.A. 0.5–1.3) or Pan Fluor 40× objective (N.A. 0.75), fit with appropriate filters and images captured with an Orca II CCD camera, model C4742–95 (Hamamatsu; Middlesex, NJ) and Metamorph image analysis and acquisition software (Molecular Devices; Sunnyvale, CA, USA). Images were exported to Photoshop (Adobe; San Jose, CA) and only linear adjustments to brightness and/or contrast were performed.

Fluorescence microscopy was performed using an IX-70 inverted epifluorescence microscope equipped with a 100x, 0.75 NA objective (Olympus), a MAC2002 shutter (Ludle), and fluorescence filter sets (Chroma, Brattleboro, VT) for Alexa 488 (490 nm band-pass excitation, 528 nm long-pass emission), and Alexa 568 (555nm band-pass excitation, 617 nm long-pass emission). Images were acquired with a ORCA II CCD camera (Hamamatsu, Bridgewater, NJ) equipped with frame grabber EDT DV PCI card controlled by Esee software (Inovision, Chapel Hill, NC). For analysis of SEP-GluN1, a cooled C4742-98 CCD camera (Hamamatsu, Bridgewater, NJ) and Metamorph imaging software in 8-bit format was used. The exposure time and gain setting were the same for all samples within a given experiment.