Immunostaining and Confocal Imaging

Cells were fixed and immunostained as described [58 (link)]. Images were acquired on a Zeiss LSM700 confocal microscopes in TIFF format. Images were arranged using Photoshop CS6 and Illustrator 6 (Adobe). Where indicated, images “masks” were created with Fiji software using maximum intensity projections of each image. For each projection, actin signals were thresholded for brightness and then converted to binary masks. Immunohistochemical staining of lungs to visualize SUM159T metastases was performed as described [7 (link)]. Briefly, formalin-fixed, paraffin-embedded tissue sections were deparaffinized in xylene and rehydrated in a series of alcohol washes. Antigen retrieval was performed by boiling slides in sodium citrate pH 6.0. Slides were blocked in 20% Aquablock (Abcam, Cambridge, MA) in TBS before being incubated overnight with the indicated antibodies, followed by species-specific secondary antibodies conjugated to AlexaFluor488, AlexaFluor546, or AlexaFluor647, and counterstained with Hoechst.