Immunofluorescence

Cells were fixed with 4% PFA in PBS and permeabilized with 0.5% Triton X-100 (Sigma) in PBS for 5 minutes. Samples were then blocked with 10% normal goat serum (KPL, Gaithersburg, USA) for 1 hour, and incubated with primary antibody against AHNAK (mouse monoclonal clone E5; Santa Cruz Biotechnology Inc., Santa Cruz, CA, USA) diluted 1:50 in PBS for 1 hour at room temperature. Primary antibody was detected by goat anti-mouse secondary antibodies, conjugated to either Alexa Fluor 568 or Alexa Fluor 647 (Life Technologies, Eugene, Oregon, USA) for 1 hour protected from light. Samples were mounted in ProLong with DAPI to stain nuclei (Life Technologies). Non-immune serum served as negative controls.

Results were analyzed by fluorescence microscopy (Axiophot, Carl Zeiss, Oberkochen, Germany) using a PlanApo 100x objective (1.45NA). Cell images were acquired with a digital CCD monochromatic camera (CoolSnap HQ2, Photometrics Inc, Tucson, AZ, USA). The microscope and additional hardware were controlled by Metamorph Premier 7.6 software (Molecular Devices, Sunnyvale, CA, USA). Samples were also analyzed by confocal microscopy with a Leica TCS AOBS SP8 Tandem Scanner with spectral detection system Leica SP Detector (Leica Microsystems, Germany). Measurements of colocalization areas were determined using ImageJ public domain software (http://rsb.info.nih.gov/ij/). Colocalization analysis was carried out by the Linescan tool (Metamorph Premier 7.6 software), and the Image J plugin JaCop (http://rsb.info.nih.gov/ij/plugins/track/jacop.html).