Plan apochromat 1.4 oil dic

HEK293T cells were seeded and transfected using Lipofectamine 2000 in six-well plates containing glass cover slips precoated with poly-L-lysine (1 mg/ml; Sigma). Forty-eight hours after transfection, cells were washed twice with PBS, fixed with 4% paraformaldehyde for 15 min, blocked in PBS-0.2% BSA and incubated for 2 h with a mouse anti-HA antibody (16B12, Covance) or a mouse anti-Myc tag (9E10, Santa Cruz). Immunoreactivity was revealed using an Oregon Green– or Texas Red–conjugated secondary goat anti-mouse antibody (Molecular Probes). Cell membranes and nuclei were respectively stained with Texas Red–conjugated wheat germ agglutinin (WGA; Molecular Probes) and DAPI (Sigma). Fluorescent images were acquired with an Axio Observer Z.1m inverted microscope associated with ApoTome (Carl Zeiss Jena, Germany) and a 63× objective lens (Plan-Apochromat, 1.4 Oil DIC). Colocalization was assessed by overlay using Zeiss microscopy software AxioVision Rel. 4.8. Confocal imaging was performed using a LSM 780 microscope, piloted by manufacturer software, with a 63× Plan-Neofluar objective (Carl Zeiss), and the colocalization parameter (correlation coefficient) was calculated for each cell using ZEN software.