Deep Imaging of Cleared Brain by Confocal Laser-Scanning Microscopy

Abstract

The Sca_l_eS technique for clearance of biological tissue enables us to perform high­ resolution three­dimensional reconstructions of entire structures in fixed, unsectioned brain samples where specific cell types are labeled with fluorescent proteins genetically or with fluorophore­conjugated antibodies. While Sca_l_eA2 and Sca_l_eU2, the previously reported urea­based clearing solutions have refractive indices (RIs) of 1.38 and 1.40, respectively, Sca_l_eS solutions containing sorbitol in addition to urea have higher RIs around 1.47. Importantly, Sca_l_eS solutions preserve fluorescence signals and tissue fine structures, allowing quantitative three­dimensional (3D) reconstructions. Here we provide a basic protocol