Fitc conjugated donkey anti mouse secondary antibody

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.

Fixation of tissues was performed with 4% paraformaldehyde in PBS for 20 min and the vessels were processed for OCT (Tissue-Tek, USA) embedding. Five μm-frozen sections were cut with a cryostat, blocked with donkey serum (BD Pharmingen, USA), and incubated in rabbit anti-mouse CD31 (1:50, Abcam, UK) and mouse anti-mouse GFP (1:50, Santa Cruz, USA) overnight at 4°C. Incubation with Cy3-conjugated donkey anti-rabbit and FITC-conjugated donkey anti-mouse secondary antibodies was carried out for 1 h (1:200, Jackson ImmunoResearch, USA) at 37°C. The slides were then treated with DAPI for 10 min at room temperature. The slides were cover-slipped and imaged with a Leica immunofluorescence microscope.