Protocol to characterize longitudinal gut motility in mice using transit time, tissue harvest, and whole-mount immunostaining

Transit time is a key in vivo metric of gastrointestinal (GI) motility, which is a physiologic readout of cellular communication within the enteric system. Here, we present a protocol to characterize longitudinal gut motility in mice. We describe steps for transit testing, whole-mount immunostaining, and tissue harvest. We then detail procedures for image processing and manual cell counting. This protocol seeks to minimize inter-trial variability while assessing cellular and molecular features that may underpin motility differences between experimental conditions. For complete details on the use and execution of this protocol, please refer to Frith et al.¹.