Cultured PDAC cells were prepared for immunofluorescence as described previously (Wang et al., 2011 (link)). Coverslips were incubated with primary antibodies in blocking buffer (5% goat serum, 5% glycerol, 0.04% sodium azide in D-PBS) overnight at 4°C, and incubated with secondary antibodies in blocking buffer for 1 h at room temperature. Alexa Fluor (Thermo Fisher) secondary antibodies were used at a concentration of 1:500. Coverslips were mounted onto glass slides using Prolong Gold (Thermo Fisher). Images were acquired using a Zeiss Axio Observer epifluorescence microscope and Zeiss LSM 780 confocal microscope (Carl Zeiss, Oberkochen, Germany) controlled by Zeiss Zen software (2012 SP1 black edition). Images processed uniformly using Adobe Photoshop software. Protein colocalization analysis was performed using the Coloc2 plug-in for ImageJ.
Live-cell imaging of GFP–α-actinin 4 and GFP-Dyn2 was performed on a Zeiss LSM 780 confocal microscope. Cells were plated into 35 mm glass-bottom imaging dishes (MatTek Corporation) before imaging. Cells were maintained in a 37°C, 5% CO2 environment over the course of imaging.
Live-cell imaging of GFP–α-actinin 4 and GFP-Dyn2 was performed on a Zeiss LSM 780 confocal microscope. Cells were plated into 35 mm glass-bottom imaging dishes (MatTek Corporation) before imaging. Cells were maintained in a 37°C, 5% CO2 environment over the course of imaging.
