A simplified and rapid in situ hybridization protocol for planarians

Abstract

Whole mount in situ hybridization is a critical technique for analyzing gene expression in planarians. While robust in situ protocols have been developed, these protocols are laborious, making them challenging to incorporate in an academic setting, reducing throughput, and increasing time to results. We systematically tested modifications to all phases of the protocol with the goal of eliminating steps and reducing time without impacting quality. Our modified protocol allows for whole mount colorimetric in situ hybridization and multicolor fluorescence in situ hybridization to be completed in two days with a significant reduction in steps and hands on processing time. Method summaryA planarian in situ hybridization protocol was optimized for time and simplicity. Modifications to the fixation, bleaching, and blocking solutions were tested to identify conditions that reduced time and number of steps without impacting staining quality.