Whole-Tissue Immunolabeling and 3D Fluorescence Imaging to Visualize Axon Degeneration in the Intact, Unsectioned Mouse Tissues

Abstract

Axon degeneration destructs functional connectivity of neural circuits and is one of the common, key pathological features of different neurodegenerative diseases. However, conventional histochemistry methods, which largely rely on tissue sections, have intrinsic limitations in examining the 3D distribution of axonal structures on the whole-tissue level. This technical shortcoming has continuously impeded our in-depth understanding of pathological axon degeneration in many scenarios. To overcome such drawback encountered in the research field, we describe here a general protocol of whole-tissue immunolabeling and 3D fluorescence imaging technique to visualize axon degeneration in the intact, unsectioned mouse tissues. In particular, experimental steps of tissue harvesting, whole-tissue immunolabeling, tissue optical clearing, and 3D fluorescence imaging have been systematically optimized, which makes the protocol effective for assessing integrity of the axonal structures in a variety of tissues. Notably, it has enabled the 3D fluorescence imaging of chemotherapy- or traumatic injury-induced axon degeneration within the bones (e.g., femurs) or bone-containing tissues (e.g., hindpaws), which had previously been inaccessible to conventional histochemistry methods. This protocol is therefore readily compatible with many areas of the research on axon degeneration and is poised to serve the field in future investigations.

Keywords: 3D fluorescence imaging; Axon degeneration; Femur; Hindpaw; Light sheet microscope; Tissue optical clearing; Whole-tissue immunolabeling.