Luo, P.Dong, J.Qi, J.Zhang, Y.Liu, X.Zhong, Y.Xian, C.J.Wang, L.2019-08-112019-08-112019BMC Neuroscience, 2019; 20(1):32-321471-22021471-2202http://hdl.handle.net/2440/120482BACKGROUND:Three-dimensional (3D) reconstruction of human peripheral nerves, as a useful tool to understand the nerve internal information and functional basis, has become an important area of research in the peripheral nerve field. METHODS:In this study, we proposed a two-dimensional (2D) Karnovsky-Roots toluidine blue ponceau 2R (K-B-2R) staining method based upon conventional Karnovsky-Roots staining. It significantly improved the ability to display nerve fascicles, motor and sensory nerve fiber textures. In this method, Karnovsky-Roots staining was carried out, followed by toluidine blue counterstain and ponceau 2R counterstain. RESULTS:Comparisons were conducted between the three methods in staining of median nerve sections, which showed similar distribution characters in acetylcholinesterase-positive sites. The additional counterstaining did not change the basis of Karnovsky-Roots staining. However, the resulting images from this new method significantly facilitated the subsequent 3D nerve reconstruction and 3D printing. CONCLUSIONS:These results show that the new staining method significantly enhanced the display qualities of nerve fascicle edges and fiber textures of motor and sensory nerves and facilitated 3D nerve reconstruction.en© The Author(s) 2019. This article is distributed under the terms of the Creative Commons Attribution 4.0 International License (http://creat iveco mmons .org/licen ses/by/4.0/), which permits unrestricted use, distribution, and reproduction in any medium, provided you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons license, and indicate if changes were made. The Creative Commons Public Domain Dedication waiver (http://creat iveco mmons .org/ publi cdoma in/zero/1.0/) applies to the data made available in this article, unless otherwise stated.Acetylcholinesterase stainingFascicle texture featureImage segmentationK-B-2RPeripheral nervesThree-dimensional reconstructionJournal article003012111910.1186/s12868-019-0515-72-s2.0-85068832065481508Xian, C.J. [0000-0002-8467-2845]