Goat anti sheep igg1 conjugated to alexa 488

Cells were grown to confluence onto glass chamber slides (Becton Dickinson Falcon) at 37°C. They were then fixed with 2% formaldehyde and 4% sucrose for 15 minutes, washed in PBS and stained with 10 μg/ml of either a sheep anti-human CDH15 antibody (Val22-Ala606) (R&D systems) or sheep IgG (Southern Biotech, UK) overnight at 4 °C. Goat anti-sheep IgG1 conjugated to Alexa 488 (1:1000) (Abcam, UK) was then applied and the slides were visualized using a Zeiss LSM 510 inverted laser scanning confocal microscope using a X40 water immersion objective with excitation at 488 nm and 543 nm (Zeiss, Gottingen, Germany). Constant acquisition parameters and laser power were maintained throughout individual experiments for analysis and images were processed using Zeiss LSM Image Examiner software (Zeiss). Digital images were recorded in two separately scanned channels with no overlap in detection of emissions from the respective fluorochromes. Confocal micrographs were stored as digital arrays of 1024×1024 pixels with 8-bit sensitivity.