利用大鼠坐骨神经分离制备单根神经纤维

Jinkun  Wen; Lixia  Li; Dandan Tan; Jiasong  Guo

doi:10.21769/BioProtoc.2572

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Peer-reviewed

Preparation of Teased Nerve Fibers from Rat Sciatic Nerve

利用大鼠坐骨神经分离制备单根神经纤维

JW Jinkun Wen

LL Lixia Li

DT Dandan Tan

JG Jiasong Guo email

发布: 2017年10月05日第7卷第19期 DOI: 10.21769/BioProtoc.2572 浏览次数: 12017

评审: Alexandros KokotosAnonymous reviewer(s)

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Cover of Molecular Neurobiology, featuring study using the protocol.

Mar 2017

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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 (外周神经)

Single nerve fiber (单神经纤维)

Tease (分离)

Myelin (髓磷脂)

Axon (轴突)

Immuno-staining (免疫染色)

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

Adhesion microscope slides (CITOTEST LABWARE MANUFACTURING, catalog number: 80312-3161-16 )
Note: These slides with special treatment process that electrostatically adheres tissue to the glass without the need for adhesives or protein coatings.
Cover slips (CITOTEST LABWARE MANUFACTURING, catalog number: 80342-1130 )
Cell culture dish (100 x 10 mm) (Corning, catalog number: 430167 )
Animal: 4-month-old Sprague Dawley (SD) rat
Chloral hydrate (Sinopharm Chemical Reagent, catalog number: 30037517 )
Sodium chloride (NaCl) (Guangdong Guanghua Sci-Tech, catalog number: 1.01307.040 )
Paraformaldehyde (PFA) (Guangdong Guanghua Sci-Tech, catalog number: 1.17767.014 )
Phosphate-buffered saline (PBS) (Beyotime Biotechnology, catalog number: C0221A )
Triton X-100 (Sigma-Aldrich, catalog number: V900502 )
Gelatin (Sigma-Aldrich, catalog number: G7041 )
Anti-S100 Protein antibody, clone 15E2E2, produced in mouse (S100) (Merck, catalog number: MAB079-1 )
Anti-Neurofilament 200 antibody produced in rabbit (NF) (Sigma-Aldrich, catalog number: N4142 )
Alexa Fluor^® 488 goat anti-mouse IgG (H+L) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A-11001 )
Alexa Fluor^® 568 goat anti-rabbit IgG (H+L) (Thermo Fisher Scientific, Invitrogen^TM, catalog number: A-11011 )
TWEEN^® 20 (Sigma-Aldrich, catalog number: P1379 )
4,6-Diamidino-2-phenylindole (DAPI) (Sigma-Aldrich, catalog number: D9542 )
Mounting medium for fluorescence (Vector Laboratories, catalog number: H-1000 )
10% chloral hydrate (see Recipes)
0.9% NaCl (see Recipes)
4% PFA (see Recipes)
0.1% Triton X-100 (see Recipes)
Blocking buffer (see Recipes)
PBST (see Recipes)
1,000x DAPI (see Recipes)

Equipment

Dissecting scissors and forceps (see Figure 1A)
Perfusion pump (Longer, catalog number: BT300-1F )
Spring scissors (66 Vision Tech, catalog number: 54053B ) (see Figure 2A)
Fine forceps (Fine Science Tools, Dumont, model: #5SF, catalog number: 11252-00 , see Figure 2A)
Black wet chamber (Leibusi, catalog number: 340012 )
Note: The wet chamber (Leibusi, catalog number: 340012 ) was made by a local workshop.
Stereomicroscope (Olympus, catalog number: SZ61 )

Procedure

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Wen, J., Li, L., Tan, D. and Guo, J. (2017). Preparation of Teased Nerve Fibers from Rat Sciatic Nerve. Bio-protocol 7(19): e2572. DOI: 10.21769/BioProtoc.2572.