Section Immunostaining for Protein Expression and Cell Proliferation Studies of Regenerating Fins.

Abstract

Adult zebrafish fins fully regenerate after resection, providing a highly accessible and remarkable vertebrate model of organ regeneration. Fin injury triggers wound epidermis formation and the dedifferentiation of injury-adjacent mature cells to establish an organized blastema of progenitor cells. Balanced cell proliferation and redifferentiation along with cell movements then progressively reestablish patterned tissues and restore the fin to its original size and shape. A mechanistic understanding of these coordinated cell behaviors and transitions requires direct knowledge of proteins in their physiological context, including expression, subcellular localization, and activity. Antibody-based staining of sectioned fins facilitates such high-resolution analyses of specific, native proteins. Therefore, such methods are mainstays of comprehensive, hypothesis-driven fin regeneration studies. However, section immunostaining requires labor-intensive, empirical optimization. Here, we present detailed, multistep procedures for antibody staining and co-detecting proliferating cells using paraffin and frozen fin sections. We include suggestions to avoid common pitfalls and to streamline the development of optimized, validated protocols for new and challenging antibodies.