Planapo n 60 1.42 na dic objective

Cells were adhered to coverslips and then treated with PEME buffer containing 1% NP-40 for 5 min. The following antibodies were used: FITC-conjugated anti-HA monoclonal antibody (Clone HA-7, 1:400 dilution), anti-HA monoclonal antibody (clone HA-7, 1:400 dilution), and anti-Protein A polyclonal antibody (1:400 dilution). All three antibodies were purchased from Sigma-Aldrich. Cells were incubated with primary antibodies at room temperature for 1 h, and then washed three times with PBS containing 0.1% Triton X-100. For anti-HA and anti-Protein A staining, cells were then incubated with Cy3-conjugated anti-mouse IgG or FITC-conjugated anti-rabbit IgG (Sigma-Aldrich, 1:400 dilution) at room temperature for 1 h. The slides were mounted in VectaShield mounting medium (Vector Labs) containing DAPI and examined using an inverted microscope (Model IX71, Olympus) equipped with a cooled CCD camera (model Orca-ER, Hamamatsu) and a PlanApo N 60× 1.42-NA DIC objective. Images were acquired and processed using the Slidebook5 software (Intelligent Imaging Innovations, Inc).