Abstract
Due to the fibrous structure of the enteric nervous system, standard microtome-based planar microscopy cannot provide an integral view of the 3-dimensional (3-D) neural networks. In this research, we integrated immunostaining, optical clearing, and confocal microscopy to develop a microtome-free method for in-depth, 3-dimensional (3-D) visualization of the mouse intestinal innervation. The pan-neuronal marker PGP9.5, the sympathetic neuronal marker tyrosine hydroxylase, and the glial marker S100□ were used as the staining targets after paraformaldehyde fixation and Triton X-100 permeabilization of the mouse ileum. The immunolabeled nerve tissues were immersed in the optical-clearing solution, FocusClearTM, to improve light transmission to facilitate deep-tissue confocal microscopy. The improved photon penetration resulting from optical clearing of the tissues led to high-resolution image acquisition of the neural network. In addition to optical clearing, tissue permeabilization by Triton X-100 was essential to increase the signal-to-noise ratio in immunostaining/imaging. We provide a convenient and straightforward method to acquire a continuous flow of the anatomic neural information in situ. This approach will change our conventional planar view of the enteric nervous system into a 3-D panorama to better understand the intestinal physiology and diseases.