Processing Insect Legs for Fluorescence Microscopy: A Method to Preserve Neuromuscular Structures for Imaging

Abstract

Source: Guan, W., et al. Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle. J. Vis. Exp. (2018).

This video describes a method to dissect, fix, and mount the Drosophila adult leg while preserving its neuromusculature intact for imaging analysis.

Protocol

This protocol is an excerpt from Guan et al., Visualize Drosophila Leg Motor Neuron Axons Through the Adult Cuticle, J. Vis. Exp. (2018).

1. Leg Dissection and Fixation

1. Take a glass multi-well plate and fill appropriate number of wells with 70% ethanol. Add 15–20 CO₂-anesthetized flies (of either sex and any age) to each well and by using a brush, gently dab the flies into the ethanol solution until flies are fully submerged.
   NOTE: This step is to remove the hydrophobicity of the cuticle. Do not wash for more than 1 min, because this increases auto-....

Results

Figure 1: Procedure to mount legs on microscope slides. Please click here to view a larger version of this figure.

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Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethanol absolute	Fisher	E/6550DF/17	Absolute analytical reagent grade
nonionic surfactant detergent	Sigma-Aldrich	T8787	Triton X-100, for molecular biology
Fine forceps	Sigma-Aldrich	F6521	Jewelers forceps, Dumont No. 5
Glass multi-well plate	Electron Microscopy Sciences	71563-01	9 cavity Pyrex, 100 mm x 85 mm
PFA	Thermofisher	28908	Pierc 16% Formaldehyde (w/v), Methanol-free
Glycerol	Fisher BioReagents	BP 229-1	Glycerol (Molecular Biology)
Spacers	Sun Jin Lab Co	IS006	iSpacer, four wells, around 12 μL working volume per well, 7 mm diameter, 0.18 mm deep
Square 22 mm x 22 mm coverslips	Fisher Scientific	FIS#12-541-B	No. 1.5-0.16 to 0.19 mm thick
Mounting Medium	Vector Laboratories	H-1000	Vectashield Antifade Mounting Medium