Novel And Efficient Method for Drosophila Heart Fluorescence Staining with Cryosectioning

Summary

The Drosophila heart sectioning and fluorescence imaging protocol simplifies studying heart structure and pathologies. This approach involves straightforward sectioning, staining, and imaging, bypassing the technical expertise needed for traditional dissection. It enhances accessibility, making Drosophila a more widely usable model for cardiac-related research within the broader scientific community.

Abstract

Drosophila heart models are widely employed in studying cardiac aging and modeling human cardiac diseases. However, the dissection of Drosophila hearts before imaging is a meticulous time-intensive process that requires advanced training and motor skills. To address these challenges, we present an innovative protocol that utilizes cryosectioning for the fluorescence imaging of Drosophila heart tissue. The protocol has been demonstrated in imaging the adult Drosophila heart but could be adapted for developmental stages. The method enhances both the efficiency and accessibility of fluorescence staining while preserving the integrity of the tissue. This protocol simplifies the process without compromising the quality of imaging, thereby reducing the dependency on technicians with highly developed training and motor skills. Specifically, we replace complex techniques, such as capillary vacuum suction, with more straightforward methods like tissue embedding. This approach allows for the visualization of cardiac structures with greater ease and reproducibility. We demonstrate the utility of this protocol by effectively detecting key cardiac markers and achieving high-resolution fluorescence and immunostaining imaging that unveils intricate details of heart morphology and cellular organization. This method provides a robust and accessible tool for researchers exploring Drosophila cardiac biology, facilitating detailed analyses of heart development, function, and disease models.

Introduction

Cardiovascular disease (CVD) is the leading cause of death globally, responsible for approximately 17.9 million deaths each year, accounting for nearly 1/3^rd of all global deaths. Drosophila melanogaster (commonly known as fruit fly) has been widely used as a model organism for studying the genetic, cellular, and molecular basis of cardiac development, physiology, metabolism, aging, and cardiomyopathies^{1,2,3,4,5,6,7}

Protocol

1. Preparation of equipment

1. Ensure that the cryostat is powered on and set to -20 °C. Allow for adequate time to elapse for the temperature to reach this point. If the machine is at ambient temperature, cooling to optimal cutting temperature can take around 5 h.
2. Power on the slide warmer or incubator, ensuring it is set to 37 °C. Ensure that slides have enough time to reach the desired temperature.
   NOTE: At this stage, labeled slides can be placed on the warmer or incubator and left indefinitely until sectioning.
3. Ensure that dry ice is ready for use. Ice can be removed from storage at – 80 °C....

Results

The method described above facilitates the study of the Drosophila heart using fluorescence imaging without tedious dissection. This is the main benefit of this method, as the conventional method of heart dissection requires the development of complex motor skills. Illustrated in Figure 1, the method is more approachable than heart dissection for new researchers and allows for experiment flexibility. Alternatively, using -80 °C storage during the OCT mold stage, specimens can b.......

Discussion

We have developed an efficient protocol for preparing a Drosophila cardiac tube for visualization using fluorescent or confocal imaging. This is preceded by a discussion of a commonly used yet time- and labor-intensive method for accessing and monitoring the cytological integrity of Drosophila heart. Our innovative and efficient method offers a concise and efficient alternative to traditional approaches by utilizing direct cryo-embedding, which preserves the structural integrity of the Drosophila

Materials

Name	Company	Catalog Number	Comments
1000 µL Pipette	Eppendorf	3123000063
1000 µL Pipette Tips	Olympus Plastics	23-165R
10X Phosphate Buffered Saline (PBS)	Fisher	J62036.K7	ph=7.4
200 Proof Ethanol	Decon Laboratories	64-17-5
20X Tris Buffered Saline	Thermo Scientific	J60877.K2	pH=7.4
Anti-Roll Glass	IMEB	AR-14047742497
Bovine Serum Albumin	Fisher	9048-46-8
Centrifuge Tubes 1.5 mL	Fisher	05-408-129
Charged Slides	Globe Scientific	1415-15
Cryosectioning Molds	Fisher	2363553
Cryostat	Leica	CM 3050 S
Cryostat Blades	C.L. Sturkey	DT554N50
Dry Ice
Fine Forceps	Fine Science Tools	11254-20
Fly Pad	Tritech Research	MINJ-DROS-FP
Hardening mounting Media with Dapi	Vectashield	H-1800
Kimwipes	Kimtech	34120
Microscope	Olympus	SZ61
Optimal Cutting Temperature Compound	Fisher	4585
Paraformaldehyde 20%	Electron Microscopy Sciences	15713
Phalloidin 594	Abnova	U0292
Razor Blades	Gravey	#40475
Spring Scissors	Fine Science Tools	15000-10
Sucrose	Fisher	S5-500