Plan neofluar 40 0.75 objective lens

Cells grown on glass coverslips and exposed to various agents were fixed with 3.7% paraformaldehyde and permeabilized with 0.25% Triton X-100. After blocking of nonspecific sites with 2.5% BSA in PBS, the cells were immunostained with antibodies to Myo1E (1/200 dilution) or to SH3P2 (1/200), and immune complexes were detected with Alexa Fluor 488– or 546–conjugated goat antibodies to rabbit IgG (1/200 dilution; Molecular Probes). F-actin was visualized with the use of Alexa Fluor 350–labeled phalloidin (Molecular Probes). Images were acquired with an Axiovert 200M microscope equipped with a FluoArc mercury lamp, a Plan-NEOFLUAR 40×/0.75 objective lens, and an AxioCam MRm camera (ZEISS). For comparison of the localization of Myo1E or of EGFP-Myo1E and its mutants, images were acquired with the same exposure time in respective experiments (typically 2 s for Myo1E and 5 s for EGFP). Images were analyzed with AxioVision software version 4.8.2 (ZEISS).

Wound healing assays were performed using serum-starved confluent cells growing on non-coated sterile cover-slides in 6-well plates without coating; a 1000-μl pipette tip was used for scratching creating an approximately 0.6 mm wide wound, as previously described in detail in Ekman et al., 2012.³⁷ (link) Photomicrographs were obtained by a microscope (Axioplan 2; Carl Zeiss MicroImaging, Inc) with a digital camera (C4742-95; Hamamatsu), using a Plan-neofluar 40×/0.75 objective lens (Carl Zeiss MicroImaging, Inc); photographs were taken at room temperature. Primary images were acquired with the camera's QED software.