Drosophila Neuromuscular Junction (NMJ) Quantification: A Method to Assess Synaptic Morphology and Function
Published: April 30, 2023
Abstract
Source: Castells-Nobau, A., et al. Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology. J. Vis. Exp. (2017).
The Drosophila neuromuscular junction (NMJ) is used as a model to study the morphology and function of synapses. This video describes important features that researchers quantify to characterize the NMJ. The example protocol demonstrates a software able to perform the quantification automatically.
Protocol
This protocol is an excerpt from Castells-Nobau et al., Two Algorithms for High-throughput and Multi-parametric Quantification of Drosophila Neuromuscular Junction Morphology, J. Vis. Exp. (2017). 1. Requirements Prior to Image Processing Perform Drosophila open book preparations of third instar wandering larvae (L3), as previously described. Co-immunolabel Drosophila NMJ terminals using a com…
Representative Results
Figure 1: Examples of Inappropriate Macro Segmentation Results. Result images after running "Drosophila NMJ Morphometrics" or "Drosophila NMJ Bouton Morphometrics". Parts of the synaptic terminal are not included in the yellow outline (A). Parts of the background are included in the synaptic terminal by the yellow outline (B). Blue skeleton line exte…
Materials
| Immunostaining | Dilution | ||
| Mouse anti-discs large 1 | Developmental Studies Hybridoma Bank | AFFN-DLG1-4D6 | 1/25 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit) |
| Rabbit anti-horseradish peroxidase | Jackson IR | 323-005-021 | 1/500 |
| Rabbit anti-Synaptotagmin | Gift from Hugo Bellen | Jan-00 | |
| Mouse anti-Cysteine string protein | Developmental Studies Hybridoma Bank | DCSP-1(ab49) | 1/10 (conjungated using the Zenon Alexa Fluor 528 Labeling Kit) |
| Mouse anti-Bruchpilot | Developmental Studies Hybridoma Bank | nc82 | Jan-50 |
| Goat anti-mouse Alexa Fluor 488 | Life technologies | A11029 | 1/200 |
| Goat anti-rabbit Alexa Fluor 568 | Life technologies | A11011 | 1/500 |
| Zenon Alexa Fluor 568 Mouse IgG1 Labeling Kit | ThermoFisher | Z25006 | |
| ProLong Gold Antifade Mountant | ThermoFisher | P36930 | |
| Equipment | |||
| Confocal microscope or fluorescence microscope | Leica SP5 | ||
| Zeiss Axio imager | |||
| Computer | Mac or Pc | ||
| Software | |||
| FIJI |
