Live Imaging of &lt;em&gt;Drosophila&lt;/em&gt; Larval Neuroblasts

Lerit, Dorothy A.; Plevock, Karen M.; Rusan, Nasser M.

doi:10.3791/51756-v

Cited by 2 publications

(3 citation statements)

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“…The first part of our protocol introduces our approach on the dissection of larval brains. As already stated 13 , a critical aspect of the preparation is to avoid damaging the brain. The most challenging aspect of this is to separate the brain from the neighboring imaginal disks without pulling excessively on the brain.…”

Section: Discussionmentioning

confidence: 97%

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Applications of Immobilization of <em>Drosophila</em> Tissues with Fibrin Clots for Live Imaging

Januschke¹,

Loyer²

2020

JoVE

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Drosophila is an important model system to study a vast range of biological questions.Various organs and tissues from different developmental stages of the fly such as imaginal discs, the larval brain or egg chambers of adult females or the adult intestine can be extracted and kept in culture for imaging with time-lapse microscopy, providing valuable insights into cell and developmental biology. Here, we describe in detail our current protocol for the dissection of Drosophila larval brains, and then present our current approach for immobilizing and orienting larval brains and other tissues on a glass coverslip using Fibrin clots. This immobilization method only requires the addition of Fibrinogen and Thrombin to the culture medium. It is suitable for highresolution time lapse imaging on inverted microscopes of multiple samples in the same culture dish, minimizes the lateral drifting frequently caused by movements of the microscope stage in multi-point visiting microscopy and allows for the addition and removal of reagents during the course of imaging. We also present custom-made macros that we routinely use to correct for drifting and to extract and process specific quantitative information from time-lapse analysis.

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Section: Discussionmentioning

confidence: 97%

“…As the manipulation of the clots can be experimentally difficult 13 to avoid excessive photodamage. We only add to these recommendations to not only maintain the brains at 25 °C during live imaging with a stage incubator, but also to preheat this stage for at least 30 min before the start of imaging.…”

Section: Discussionmentioning

confidence: 99%

Applications of Immobilization of <em>Drosophila</em> Tissues with Fibrin Clots for Live Imaging

Januschke¹,

Loyer²

2020

JoVE

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“…For this study, age‐matched larval brains were identified by partial gut clearance, dissected, fixed, and stained for DAPI as a marker for brain volume (Lerit et al., 2014; Link et al., 2019). Entire brain volumes were then imaged on a spinning disk confocal microscope.…”

Section: Commentarymentioning

confidence: 99%

Colorimetric Synchronization of Drosophila Larvae

Hailstock,

Terry,

Wardwell‐Ozgo

et al. 2023

Current Protocols

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The rapid succession of events during development poses an inherent challenge to achieve precise synchronization required for rigorous, quantitative phenotypic and genotypic analyses in multicellular model organisms. Drosophila melanogaster is an indispensable model for studying the development and function of higher order organisms due to extensive genome homology, tractability, and its relatively short lifespan. Presently, nine Nobel prizes serve as a testament to the utility of this elegant model system. Ongoing advancements in genetic and molecular tools allow for the underlying mechanisms of human disease to be investigated in Drosophila. However, the absence of a method to precisely age‐match tissues during larval development prevents further capitalization of this powerful model organism. Drosophila spends nearly half of its life cycle progressing through three morphologically distinct larval instar stages, during which the imaginal discs, precursors of mature adult external structures (e.g., eyes, legs, wings), grow and develop distinct cell fates. Other tissues, such as the central nervous system, undergo massive morphological changes during larval development. While these three larval stages and subsequent pupal stages have historically been identified based on the number of hours post egg‐laying under standard laboratory conditions, a reproducible, efficient, and inexpensive method is required to accurately age‐match larvae within the third instar. The third instar stage is of particular interest, as this developmental stage spans a 48‐hr window during which larval tissues switch from proliferative to differentiation programs. Moreover, some genetic manipulations can lead to developmental delays, further compounding the need for precise age‐matching between control and experimental samples. This article provides a protocol optimized for synchronous staging of Drosophila third instar larvae by colorimetric characterization and is useful for age‐matching a variety of tissues for numerous downstream applications. We also provide a brief discussion of the technical challenges associated with successful application of this protocol. © 2023 Wiley Periodicals LLC.Basic Protocol: Synchronization of third instar Drosophila larvae

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Live Imaging of <em>Drosophila</em> Larval Neuroblasts

Cited by 2 publications

References 0 publications

Applications of Immobilization of <em>Drosophila</em> Tissues with Fibrin Clots for Live Imaging

Applications of Immobilization of <em>Drosophila</em> Tissues with Fibrin Clots for Live Imaging

Colorimetric Synchronization of Drosophila Larvae

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