Fluorescence Photoactivation Imaging of Neurofilament Transport in Myelinated Mouse Axons

Abstract

Source: Boyer, N. P. et. al., Imaging and Analysis of Neurofilament Transport in Excised Mouse Tibial Nerve. J. Vis. Exp. (2020)

This video demonstrates the fluorescence photoactivation method to analyze neurofilament transport in myelinated axons of peripheral nerves. By tracking photoactivated green fluorophore-tagged neurofilaments, the technique provides insights into axonal transport in neurodegenerative diseases.

Protocol

All procedures involving animal models have been reviewed by the local institutional animal care committee and the JoVE veterinary review board.

Results

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
14 x 22 Rectangle gasket 0.1mm	Bioptechs	1907-1422-100	Inner gasket
2-deoxy-D-glucose	Sigma	D6134
30mm Round gasket w/ Holes	Bioptechs	1907-08-750	Outer gasket
35 x 10mm dish	Thermo Fisher	153066	Dissection dishes
40mm round coverslips	Bioptechs	40-1313-0319
60mL syringe - Luer-lock tip	BD	309653
Andor Revolution WD spinning-disk confocal system	Andor		Outfitted with Perfect Focus and FRAPPA systems
Calcium chloride	Fisher	C79
Coverslips	Fisher	12-541-B	For fluorescein slide
D-(+)-glucose solution	Sigma	G8769
Dissecting pins	Fine Science Tools	26001-70
Dissection forceps	Fine Science Tools	11251-30	Fine tipped forceps
Dissection microscope	Zeiss	47 50 03
Dissection pan with wax	Ginsberg Scientific	568859
Dissection scissors	Fine Science Tools	14061-09	Initial dissection scissors
FCS2 perfusion chamber	Bioptechs	60319-02-03
Fluorescein sodium	Fluka	46960
Inline solution heater	Warner Instruments	SH27-B
Laminectomy forceps	Fine Science Tools	11223-20	Initial dissection forceps
Magnesium sulfate	Sigma-Aldrich	M7506
Microaqueduct slide	Bioptechs	130119-5
Microscope slides	Fisher	12-544-3	For fluorescein slide
Microscope stage insert	Applied Scientific Instrumentation	I-3017
Objective heater system	Okolab		Oko Touch with objective collar
Objective oil - type A	Nikon	discontinued
Plan Apo VC 100x 1.40 NA objective	Nikon	MRD01901
Potassium chloride	Fisher	P217
Potassium phosphate	Sigma-Aldrich	P0662
Sodium bicarbonate	Sigma-Aldrich	S6297
Sodium chloride	Sigma-Aldrich	S7653
Syringe pump	Sage Instruments	Model 355
Tubing adapter - female	Small Parts Inc.	1005109
Tubing adapter - male	Small Parts Inc.	1005012
Tygon tubing	Bioptechs		1/16" ID, 1/32" wall thickness
Vannas spring scissors	Fine Science Tools	15018-10	fine scissors