Processing Insect Legs for Fluorescence Microscopy: A Method to Preserve Neuromuscular Structures for Imaging
Published: April 30, 2023
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 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…
Representative Results
Figure 1: Procedure to mount legs on microscope slides. Please click here to view a larger version of this figure.
Materials
| 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 |
Cite This Article
Processing Insect Legs for Fluorescence Microscopy: A Method to Preserve Neuromuscular Structures for Imaging. J. Vis. Exp. (Pending Publication), e20121, doi: (2023).