To study the compatibility of traditional tracers and viral tracers with tissue clearing technology and to provide guidance in choosing suitable tracing methods for a specific tissue clearing technique. Experiment 1: In this study, two different types of representative tracers, namely fluorescent dye tracers (Fluoro-Gold and Fluoro-Ruby) and viral tracers carrying fluorescent proteins (rAAV9-hSyn-mCherry-WPRE-pA and rAAV9-hSyn-EGFP-WPRE-pA), were selected to trace the cerebrospinal tract of the animals by microinjection. Furthermore, we presented the signal changes after using the three representative transparentizing methods, which included FRUIT (aqueous tissue clearing), 3DISCO (solvent-based tissue clearing), and uDISCO (solvent-based tissue clearing), were compared after slicing. Experiment 2: Based on the research mentioned above, Fluoro-Ruby was microinjected unilaterally into the primary motor cortex of rats, directly tracing the pyramidal tract to the spinal cord. Then, the entire brain and spinal cord were collected for tissue transparency using the 3DISCO method, after which three-dimensional imaging was performed using optical microscopic imaging equipment. Experiment 1 indicated that Fluoro-Gold and Fluoro-Ruby displayed better compatibility with the three transparent methods. The viral tracer exhibited higher compatibility with the FRUIT method, while its compatibility with 3DISCO and uDISCO was low. Furthermore, GFP was quenched more quickly and seriously than cherry protein under the same experimental conditions. Experiment 2: The Fluoro-Ruby tag displayed the presence of long-distance axons. For microscopic imaging, light sheet microscopy and two-photon microscopy were both used to identify the signals of tracers in transparent tissue. Both Fluoro-Gold and Fluoro-Ruby displayed excellent compatibility with tissue clearing technology, which, with dehydration and delipidation at its core, lead to quenching of fluorescence proteins, while exhibiting poor compatibility with viral tracers. In combination with tissue clearing technology and optical microscopy, the anterograde tracer Fluoro-Ruby could stereoscopically display the complete neural conduction pathway. Copyright © 2020 Elsevier B.V. All rights reserved.