Multiple Immunofluorescence, 3D Visualization and Quantification in Cleared Whole Mouse Lungs: Pulmonary Neuroepithelial Bodies Contacted by Myelinated Vagal Afferents

Neuroepithelial bodies (NEBs) are clusters of pulmonary neuroendocrine cells (PNECs) that represent less than one percent of the airway epithelial cells. PNECs store and release neurotransmitters, are surrounded by a unique population of stem cells, and are contacted by extensive nerve terminals. Today, multidisciplinary approaches allow combining functional morphological investigations in cryosections, live cell imaging in lung vibratome slices and selective gene expression analysis after laser microdissection of genetically tagged NEBs. However, due to their relatively low number and widespread distribution, the information remains highly fragmentary and quantification of NEBs, associated nerve endings and stem cells is problematic. The present study aimed at acquiring the first whole lung visualization and quantification of the total population of NEBs. As a proof‐of‐concept, we used a challenging triple immunolabeling and four channel imaging protocol for simultaneous identification of the selective myelinated vagal sensory innervation of NEBs. Lungs of GAD67‐GFP mice, which harbor GFP fluorescent NEB cells, were subjected to multiple immunostaining and tissue clearing. The list of optical clearing techniques is quickly growing, and several were tested; for our goals the iDisco+ protocol appeared to result in both the most complete clearing of mouse lungs and the best preservation of immunofluorescence in NEB nerve fiber populations. After fixation, methanol treatment, and permeabilization, whole mouse lungs were simultaneously immunostained for GFP (NEB cells), calbindin D‐28k and P2X3 ATP receptors (selective markers for two subpopulations of myelinated vagal afferents in mouse NEBs), and myelin basic protein (marker for all myelinated nerve fibers). Immunostained whole lungs could be imaged using light sheet microscopy (Ultramicroscope II, LaVision Biotec; Olympus MVPLAPO 2× objective; lasers: 488, 561, 640 and 785nm; emission filters: 525/50, 620/60, 680/30 and 845/55nm; Andor Neo sCMOS camera) at a total magnification of 1.26× and a z‐step of 10 μm. Optical sections were recorded in a mosaic of two by two tiles, and images stitched and merged with a linear blending algorithm (ImageJ). It could be shown that the applied staining, clearing and imaging protocols allow for 3D rendering of the complete lungs, with all NEBs and associated myelinated nerve terminals, including possibilities for 3D quantification. Selected individual NEB/nerve terminal complexes can be further analyzed in detail using high resolution confocal imaging and 3D reconstruction (Leica TCS SP8 DLS). The presented methodology opens interesting opportunities for imaging and quantification of total populations of unevenly dispersed microscopic structures in whole lungs, even when visualization requires complex immunostaining. Future goals include dedicated automated 3D quantification and shortening of the protocol time, which for now is about four weeks. Support or Funding Information Support: UA grant GOA BOF 2015 (30729 to DA).