Quantifying Cell Proliferation Through Immunofluorescence on Whole-Mount and Cryosectioned Regenerating Caudal Fins in African Killifish

The African killifish Nothobranchius furzeri is an attractive research organism for regeneration- and aging-related
studies due to its remarkably short generation time and rapid aging. Dynamic changes in cell proliferation are an
essential biological process involved in development, regeneration, and aging. Quantifying the dynamics of cell
proliferation in these contexts facilitates the elucidation of the attendant underlying mechanisms. Whole-mount and
cryosectioning sample preparation are the preferred approaches to investigate the distribution of cellular structures,
cell–cell communication, and spatial gene expression within tissues. Using African killifish caudal fin regeneration
as an example, we describe an efficient and detailed protocol to investigate cell proliferation dynamics in both space
and time during caudal fin regeneration. The quantification of cell proliferation was achieved through high-resolution
immunofluorescence of the proliferation marker Phospho-Histone H3 (H3P). We focused on the characterization of
epithelial and mesenchymal proliferation in three-dimensional space at two regeneration time points. Our protocol
provides a reliable tool for comparing cell proliferation under different biological contexts.