Fluorescent Labeling of Drosophila Heart Structures

Summary

Here we describe a basic protocol for fluorescent labeling of different elements of heart tubes from larva and adult Drosophila melanogaster. These specimens are well-suited for imaging via fluorescent or confocal microscopy. This technique permits detailed structural analysis of the features of the hearts from a powerful model organism.

Abstract

The Drosophila melanogaster dorsal vessel, or heart, is a tubular structure comprised of a single layer of contractile cardiomyocytes, pericardial cells that align along each side of the heart wall, supportive alary muscles and, in adults, a layer of ventral longitudinal muscle cells. The contractile fibers house conserved constituents of the muscle cytoarchitecture including densely packed bundles of myofibrils and cytoskeletal/submembranous protein complexes, which interact with homologous components of the extracellular matrix. Here we describe a protocol for the fixation and the fluorescent labeling of particular myocardial elements from the hearts of dissected larvae and semi-intact adult Drosophila. Specifically, we demonstrate the labeling of sarcomeric F-actin and of α-actinin in larval hearts. Additionally, we perform labeling of F-actin and α-actinin in myosin-GFP expressing adult flies and of α-actinin and pericardin, a type IV extracellular matrix collagen, in wild type adult hearts. Particular attention is given to a mounting strategy for semi-intact adult hearts that minimizes handling and optimizes the opportunity for maintaining the integrity of the cardiac tubes and the associated tissues. These preparations are suitable for imaging via fluorescent and confocal microscopy. Overall, this procedure allows for careful and detailed analysis of the structural characteristics of the heart from a powerful genetically tractable model system.

Protocol

Before you start

1. Prepare the following solutions:
   1. relaxing buffer (artificial Drosophila hemolymph (ADH) (see “Visualizing the beating heart in Drosophila”) that contains 10 mM EGTA)
   2. fixative (4% formaldehyde in 1x PBS)
   3. PBSTx (PBS containing 0.1% Triton-X-100)
   4. Appropriately diluted primary and species-specific fluorescently labeled secondary antibodies in PBSTx
2. Dissect Drosophila to expose the cardiac tubes (of adult flies and/or larvae) following
   1. the semi-intact Droso....

Discussion

Here we present a protocol useful for preparing and staining the Drosophila melanogaster dorsal vessel and associated tissues for imaging via fluorescent or confocal microscopy. We provide a concise account of the steps refined by and commonly employed in our lab for effective staining that permits well-resolved in situ imaging of larval and adult Drosophila heart tubes. Others have described similar methods in abbreviated form^{2, 3, 4}.

Important additiona.......

Materials

List of materials used in this article

Name	Company	Catalog Number	Comments
Ethylene glycol-bis (2-amino-ethylether) -N,N,N’,N’-tetra-acetic acid (EGTA)	Reagent	Sigma-Aldrich	E4378
Formaldehyde, 10%, methanol free, Ultra Pure	Reagent	Polysciences, Inc.	50-00-0
Triton-X-100	Reagent	Sigma-Aldrich	9002-93-1
Alexa Fluor® 594 phalloidin	Reagent	Invitrogen	A12381
Vectashield® Mounting Medium for Fluorescence with DAPI	Reagent	Vector Laboratories	H-1200
Tungsten pins	Reagent	Fine Science Tools	26002-10
Pin holder	Reagent	Fine Science Tools	26018-17