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

## TL;DR

This paper provides a detailed protocol for studying gut motility in mice using transit time, tissue analysis, and immunostaining to understand cellular communication in the gut.

## Contribution

A reproducible protocol for assessing gut motility and analyzing cellular features in mice with minimized variability.

## Key findings

- The protocol includes transit testing, tissue harvest, and whole-mount immunostaining for gut motility studies.
- Image processing and manual cell counting are detailed to assess cellular and molecular features.
- The method aims to reduce inter-trial variability in motility assessments.

## Abstract

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

•Guidance for reproducible in vivo gastrointestinal motility studies in mice•Workflow for gastrointestinal tissue harvest for multiple downstream applications•Whole-mount staining of enteric neurons and glia for confocal imaging of myenteric ganglia

Guidance for reproducible in vivo gastrointestinal motility studies in mice

Workflow for gastrointestinal tissue harvest for multiple downstream applications

Whole-mount staining of enteric neurons and glia for confocal imaging of myenteric ganglia

Publisher’s note: Undertaking any experimental protocol requires adherence to local institutional guidelines for laboratory safety and ethics.

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.