Metavue imaging software

Confluent control or SUSD3 siRNA-transfected MCF7 cells in 6-well plates were washed with PBS and incubated with 2.4U/ml dispase (Roche) for 30min at 37°C. Released monolayers were fixed by formalin and fragments were counted using an MZ6 dissecting scope (Leica, Germany) as described previously^{56 (link)} imaged with a Hamamatsu Orca digital camera and analyzed using MetaVue imaging software (Universal Imaging, Downingtown, PA). Under experimental conditions where fragmentation was excessive, a maximum of 400 fragments was counted.

SH-SY5Y cells were plated onto cover slips and after differentiation and treatments, they were fixed in PBS containing 4% paraformaldehyde without prior permeabilization with detergent in a similar manner as previously reported (Yang et al., 2009 , 2010 (link)). After washing three times with PBS, nonspecific binding of antibodies was blocked by 5% goat serum for 1 h at room temperature. Cells were then incubated overnight at 4 °C in 3% goat serum with anti-APP mouse antibody (1:200 dilution; assay designs, Ann Arbor, MI, USA) that recognizes the N-terminus of APP. The cover slips were washed with PBS and incubated for 1 h at room temperature with fluorescein isothiocyanate (FITC)-labeled goat anti-mouse secondary antibody (1:400, Santa Cruz, CA, USA) and washed with PBS. Cover slips were then mounted and fluorescent intensity measurements were performed at room temperature using the Nikon TE-2000 U fluorescence microscope and oil immersion 60× objective lens. Images were acquired using a CCD camera controlled by a computer running a MetaVue imaging software (Universal Imaging, PA, USA). The fluorescent intensities per cell area were measured. Background subtraction was done for all images prior to data analysis.