Optimized Fixation and Phalloidin Staining of Basally Localized F-Actin Networks in Collectively Migrating Follicle Cells

Abstract

The follicular epithelial cells of the Drosophila egg chamber have become a premier model to study how cells globally orient their actin-based machinery for collective migration. The basal surface of each follicle cell has lamellipodial and filopodial protrusions that extend from its leading edge and an array of stress fibers that mediate its adhesion to the extracellular matrix; these migratory structures are all globally aligned in the direction of tissue movement. To understand how this global alignment is achieved, one must be able to reliably visualize the underlying F-actin; however, dynamic F-actin networks can be difficult to preserve in fixed tissues. Here, we describe an optimized protocol for the fixation and phalloidin staining of the follicular epithelium. We also provide a brief primer on relevant aspects of the image acquisition process to ensure high quality data are collected.