Open AccessImmunofluorescence and image analysis pipeline for Drosophila motor neurons
Author(s) -
Jeremy Brown,
Chanpasith Phongthachit,
Mikolaj J. Sulkowski
Publication year - 2019
Publication title -
biology methods and protocols
Language(s) - English
Resource type - Journals
SCImago Journal Rank - 0.555
H-Index - 5
ISSN - 2396-8923
DOI - 10.1093/biomethods/bpz010
Subject(s) - immunofluorescence , pipeline (software) , drosophila (subgenus) , indirect immunofluorescence , image (mathematics) , anatomy , neuroscience , microbiology and biotechnology , biology , artificial intelligence , computer science , engineering , genetics , gene , antibody , mechanical engineering
The neuromuscular junction (NMJ) of larval Drosophila is widely used as a genetic model for basic neuroscience research. The presynaptic side of the NMJ is formed by axon terminals of motor neurons, the soma of which reside in the ventral ganglion of the central nervous system (CNS). Here we describe a streamlined protocol for dissection and immunostaining of the Drosophila CNS and NMJ that allows processing of multiple genotypes within a single staining tube. We also present a computer script called Automated Image Analysis with Background Subtraction which facilitates identification of motor nuclei, quantification of pixel intensity, and background subtraction. Together, these techniques provide a pipeline for neuroscientists to compare levels of different biomolecules in motor nuclei. We conclude that these methods should be adaptable to a variety of different cell and tissue types for the improvement of efficiency, reproducibility, and throughput during data quantification.