Protocol for combining live imaging and in situ hybridization chain reaction to evaluate differential neutrophil interactions in zebrafish larvae

Oncogenic HRAS drives preneoplastic cell (PNC) initiation in basal skin keratinocytes, which recruit neutrophils. Here, we present a protocol for combining in vivo time-lapse imaging of zebrafish larvae with immediate fixation and hybridization chain reaction (HCR) hybridization to identify PNC clone subtypes. We describe steps for tracking and quantifying neutrophil behaviors toward defined PNC subclusters. This protocol is adaptable to similar settings where heterogeneous target cells recruit neutrophils and post-imaging marker labeling is required to determine target cell identity. For complete details on the use and execution of this protocol, please refer to Eliott et al. 1 1. Eliott, A.M. ∙ Bravo, I.R. ∙ Zhao, Y. ... Oncogenic Ras activation in permissive somatic cells triggers rapid-onset phenotypic plasticity and elicits a tumor-promoting neutrophil response Cell Rep. 2025; 44 , 116478 Google Scholar