Protocol for spatial quantitative analysis of cell division events across the epidermis of developing Hibiscus trionum petals

Characterizing cell proliferation across developing tissue provides critical insights into how spatially regulated growth shapes mature organs. Here, we present a protocol for high-resolution mapping of cell division events across the epidermis of Hibiscus trionum petals. We outline steps for labeling newly replicated DNA through incubation in EdU-containing medium, combined with floral bud dissection and confocal imaging. We also detail procedures for sample clearing and labeling of nuclei and cell outlines to enable accurate quantification of division patterns. For complete details on the use and execution of this protocol, please refer to Riglet et al. 1 • Protocol to visualize and quantify cell division events across the petal epidermis • Steps for petal dissection, clearing, labeling, and confocal imaging • Guidance for data processing to extract the position of Edu-labeled nuclei • Provide R scripts to compare distribution of cell division events between samples Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics. Characterizing cell proliferation across developing tissue provides critical insights into how spatially regulated growth shapes mature organs. Here, we present a protocol for high-resolution mapping of cell division events across the epidermis of Hibiscus trionum petals. We outline steps for labeling newly replicated DNA through incubation in EdU-containing medium, combined with floral bud dissection and confocal imaging. We also detail procedures for sample clearing and labeling of nuclei and cell outlines to enable accurate quantification of division patterns.