Abstract
Compared to tissue sectioning techniques, the technique of teasing single nerve fibers provides a better way to understand the structures of myelin sheaths and axons of the peripheral myelinated nerves. This protocol describes a method for preparation of teased single nerve fibers from rat sciatic nerve. In this protocol, fixed nerves are teased into single individual fibers and arranged onto adhesion microscope slides for further immuno-staining.
Keywords: Peripheral nerve, Single nerve fiber, Tease, Myelin, Axon, Immuno-staining
Background
Schwann cells in the peripheral nervous system wrap around axons to form insulated myelin sheaths that allow the rapid conduction of action potentials. The remarkable multi-layered myelin sheath consists of elaborate structures including compact sheath, Schmidt-Lanterman incisures, Cajal bands, inner and outer mesaxons, as well as the structures in the paranodal region. To elucidate the normal and abnormal structures of myelinated fibers of peripheral nerves, teased nerve fibers are required. Methods of teasing fibers have been widely applied in studies on peripheral nerves of human and rodents. In this protocol, we describe a method of teasing peripheral nerves into single fibers for further morphological studies on the axons or myelin of peripheral nerves.
Materials and Reagents
Equipment
Procedure
Data analysis
Representative immunofluorescent stained images to show the structures of teased nerve fibers in Figure 3. Figure 3. Representative immunofluorescent stained images to show the structures of teased nerve fibers. A. A single individual nerve fiber teased from the sciatic nerve of 2-week old rats was double stained with S100 (a specific marker expressed in the cytoplasm of Schwan cells) and neurofilament (NF, a specific marker for showing axon). B. A single individual nerve fiber teased from the sciatic nerve of 4-month old rats was immunostained with S100 to show the detailed structures of Cajal bands (CB) and Schmidt-Lanterman incisures (SLI) which are only exist in adult nerve fibers. PNC, perinuclear cytoplasm; NR, node of Ranvier. Note: Both of images were stained with DAPI to show the nuclei of Schwann cells.
Recipes
Acknowledgments
This protocol is adapted from the previously published paper (Wen et al., 2017). This work was supported by the National Key Basic Research Program of China (2014CB542202 and 2014CB542205), National Natural Science Foundation of China (30973095, 81371354 & 81571182); Science and Technology Project of Guangzhou (12C32121609) and Natural Science Foundation of Guangdong Province (S2013010014697) to J Guo.
References
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