This protocol explains how viscerofugal neurons can be studied in ex vivo specimens of human colon. Viscerofugal neurons are characterized by having cell bodies in myenteric ganglia and axons that projectout of the gut via colonic or mesenteric nerves. The protocol covers obtaining and handling live human colonic tissue specimens, biotinamide tracing of viscerofugal neuron cell bodies from axons in colonic nerves, tissue culture, fixation and immunohistochemical processing. This protocol was used in: Chen BN, Humenick A, Hibberd TJ, Yew WP, Wattchow DA, Dinning PG, Costa M, Spencer NJ, Brookes SJH (2024) Characterisation of viscerofugal neurons in human colon by retrograde tracing and multi-layer immunohistochemistry.Frontiers in Neuroscience 17:1313057. doi: 10.3389/fnins.2023.1313057
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