Retrograde Labeling of Drosophila Motor Neurons Using Fluorescent Lipophilic Dyes

Abstract

Source: Inal, M. A. et. al., Retrograde Tracing of Drosophila Embryonic Motor Neurons Using Lipophilic Fluorescent Dyes. J. Vis. Exp. (2020)

This video demonstrates the method of retrograde labeling of Drosophila motor neurons using lipophilic dyes. The dye enters the axon, travels retrogradely, and labels the neurons green, allowing detailed visualization of their morphology and projections under a confocal microscope.

Protocol

1. Equipment and Supplies

1. Materials for collecting embryos and training adults to lay eggs
   1. Prepare the filtration apparatus by severing a 50 mL tube and cutting open a hole in the cap to set a mesh filter with pores of 100 µm (Table of Materials) in between the tube and the cap.
      NOTE: Alternatively, cell strainers with pores of 10....

Results

Figure 1: Setup of the dissection pool. The blue chamber seen on the glass slide is created wit.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
10x objective lens	Nikon		Plan
40x water-immersion lens	Nikon		NIR Apo
Capillary tubing	Frederick Haer&Co	27-31-1
Confocal microscope	Andor	N/A	Dragonfly Spinning disk confocal unit
Cover glass	Corning		22x22 mm Square #1
DiD	ThermoFisher	V22886
DiI	ThermoFisher	V22888
DiO	ThermoFisher	V22887
Dissecting microscope	Nikon	N/A	SMZ-U
Double sided tape	Scotch	665
Dow corning high-vacuum grease	Fisher Sci.	14-635-5D
Dumont #5 Forceps	Fine Science Tools	11252-20
Egg collection cage	FlyStuff	59-100
FemtoJet 5247	Eppendorf	discontinued	FemtoJet 4i (Cat No. 5252000021)
Micromanipulator	Sutter	MP-225
Micropipette beveler	Sutter	BV-10-B
Needle puller	Narishige	PC-100
Nutri-fly grape agar powder premix packets	FlyStuff	47-102
Nylon net filter	Millipore
Paraformaldehyde 16% solution, EM grade	Electron Microscopy Sciences	15710	Any EM grades
PBS	Roche	11666789001	Sold on sigmaaldrich, boxed 10x solution
Photo-Flo 200	Kodak	146 4510	Wetting agent
Upright fluorescence microscope	Nikon	N/A	Eclipse Ci with a LED light source
Vinyl electrical tape	Scotch	6143
VWR Cell strainers	VWR	10199-659
Yeast	FlyStuff	62-103	Active dry yeast (RED STAR)