Labeled secondary antibodies

Cells were fixed and prepared for immunofluorescence as described previously (Wang et al., 2011 (link)). Coverslips were incubated in primary antibodies for 2 h at 37°C. The primary antibodies used were FLAG (Cell Signaling), MT1-MMP (Abcam), and Tks5 (Santa Cruz). Then the coverslips were washed with d-PBS and incubated in labeled secondary antibodies (Life Technologies) for 1 h at room temperature. Actin was visualized by TRITC-phalloidin or Phalloidin-Atto 390 (Sigma Aldrich) staining. Prolong mounting medium (Life Technologies) was used to mount the coverslips before imaging. Fluorescence images were acquired using epifluorescence microscopes (Axio Observer and Axiovert 200; Carl Zeiss MicroImaging) using a 63× oil objective, unless otherwise indicated, with iVision software. For confocal microscopy, cells were imaged with a Zeiss LSM 780 confocal microscope (Carl Zeiss MicroImaging) and a 63×/1.2-NA water immersion lens. Images were processed and adjusted with Adobe Photoshop software (Adobe) uniformly to the entire image.

Cells were prepared for immunofluorescence as described previously.⁽²²⁾ Briefly, cells were incubated with primary antibodies for 2 hours at 37°C, washed with D‐PBS, and incubated with labeled secondary antibodies (Life Technologies, Carlsbad, CA) for 1 hour at 37°C. Actin was stained using TRITC‐Phalloidin, and the nucleus was stained with 4′,6‐diamidino‐2‐phenylindole (DAPI; Sigma‐Aldrich). The coverslips were mounted on glass slides using ProLong mounting medium (Life Technologies), and cells were imaged with an AxioObserver D.1 epifluorescence microscope (Carl Zeiss, Thornwood, NY) equipped with a 100‐W mercury lamp using a × 63, 1.4N.A objective lens. Contrast and intensity for each image were manipulated uniformly using Adobe Photoshop software (Adobe Systems Inc., San Jose, CA). Images were analyzed with the IVision software (BioVision, Mountain View, CA).