Fish epidermal keratocytes provide a powerful model system for investigating cell motility due to their rapid, persistent migration and stable morphology. While single-cell dynamics have been extensively characterized, recent studies have increasingly focused on collective migration. Because keratocytes cannot be maintained as continuous cell lines, experiments rely on primary cultures derived from fish scales. This chapter presents optimized protocols for culturing primary keratocytes, transiently introducing fluorescent probes using a custom electroporation system and performing high-resolution three-dimensional time-lapse imaging with confocal microscopy. These procedures enable visualization of dynamic molecular and cellular behaviors in both single-cell and collective migration contexts.