Movie 3: 3D visualization of calcitonin gene-related peptide (CGRP) and substance P (SP) positive varicosities around a neuron within the superior hypogastric plexus (SHP)

A sample block of adult human (male, 22 years) SHP was preserved by paraformaldehyde (PFA) fixation (unembalmed) and prepared for immunofluorescence. Cryosections (50 µm) of the SHP were stained with CGRP and SP to investigate putative visceral afferent fibers. A Z-stack of the neuron and associated CGRP- and SP-immunoreactive varicosities was captured with confocal microscopy. Manual segmentation of the neuronal cell body (white) using autofluorescence (captured in the 488 nm channel) was completed using the Surfaces tool in Imaris. Semi-automated segmentation of CGRP (green) and SP (magenta) varicosities show their density, arrangement and colocalization around the neuron in a three-dimensional view.Human tissue specimens were obtained from the organ donation program based at the Centre Hospitalier Universitaire de Nîmes (CHU de Nîmes), France and the Body Donor Program at the University of Melbourne, Australia. Ethical approval for the use of human tissue was granted from Human Research Ethics Committees at both institutions.