Immunofluorescence Imaging of AnxA2 in PC12 Cells

PC12 cells were grown on poly-L-Lys-coated glass coverslips and treated as indicated above. The cells were fixed, permeabilised and blocked against non-specific binding of antibodies as described previously (Grindheim et al., 2014 (link); Grindheim et al., 2016 (link)) prior to staining with primary polyclonal antibodies against AnxA2 (1:250; ab41803, Abcam, Cambridge, UK). The bound primary antibodies were detected using appropriate DyLight-488- or DyLight-594-conjugated Fab2 fragments (1:50; Jackson ImmunoResearch Laboratories, West Grove, United States). The coverslips were inverted and mounted on objective glasses on a small drop of Vectashield mounting medium containing 4′,6-diamino-2′-phenylindole (DAPI) (Vector Laboratories, Burlingame, United States). Confocal imaging was performed using a Leica SP5 AOBS confocal laser scanning microscope equipped with 405 diode and argon and helium neon lasers (Leica Microsystems, Wetzlar, Germany). Optical sections were obtained using the 63×/1.4 NA HCX Plan-Apochromat oil-immersion objective (Leica, Wetzlar, Germany), ∼1 Airy unit pinhole aperture and appropriate filter combinations. Confocal images were obtained in Leica Application Suite (LAS) AF. Figures were made in Microsoft Publisher for the images and GraphPad Prism for the graphs. Quantitation was done with ImageJ and transferred to GraphPad.