Novel And Efficient Method for Drosophila Heart Fluorescence Staining with Cryosectioning

My research lab uses drosophila model to study aging and metabolic disorder, exploring time-restricted feeding and exercise as intervention for metabolic, cardiac muscle, sleep, and other aging diseases. Machine learning and omic with physiological genetic and nutritional strategy is streamlining drosophila assay using 3D-printed devices and machine learning approaches. Fluorescent immunohistochemistry of drosophila hearts enables multi-protein detection across genotypes and ages, but faces challenges like complex dissections, tissue preservation, and high-resolution imaging of a specific protein expression patterns.

This protocol improves tissue preservation, antibody penetration, and imaging resolution, enabling better visualizations of cardiac protein markers. It addresses challenges like complex dissection, tissue damage, and limited resolution in traditional whole-heart mount method. This protocol enhances drosophila heart research accessibility by eliminating complex dissection, enabling one-handed execution, and avoiding costly confocal microscopy, making high resolution imaging more feasible.

To begin, anesthetize 5 to 10 flies using a carbon dioxide fly mat. Position the flies within view under the microscope at 1X to 2X magnification. Using spring scissors, decapitate the flies, clip the wings, and remove the legs.

Retain the thorax and abdomen together, referred to as the body. Using a brush, gently placed the bodies into labeled 1.5-milliliter tubes on ice until all experimental groups have been collected. After removing the tubes from the ice, add 100 microliters of 10-millimolar ethylene glycol tetraacetic acid at pH 8 to each tube.

Ensure all bodies are submerged and place the tubes on an orbital shaker for 10 minutes. Then, remove and discard the ethylene glycol tetraacetic acid solution from the tubes. Pipette 100 microliters of PBS into each tube to wash the tissue for 10 minutes.

Place the tubes on the orbital shaker during the washing process. After discarding the PBS, pipette 100 microliters of 4%paraformaldehyde in PBS solution into each tube. Place the tubes on an orbital shaker at 100 units for 15 minutes, ensuring the tissues remain in contact with the solution.

Then, discard the paraformaldehyde solution. Once the final wash is complete, transfer the bodies to a solution of 10%sucrose in PBS. Ensure the bodies are in contact with the solution within the tube.

Retrieve dry ice from storage and select a few pieces for placement in a small metal or plastic tray. Choose a tray with dimensions that allow the mold to be inserted and place it directly on top of a single large and flat piece of dry ice. Fill a labeled mold to 50%with optimal cutting temperature, or OCT compound, pouring slowly to minimize bubbles.

With a brush, carefully place the dissected and fixed drosophila bodies on the surface of the OCT compound within the mold. Using forceps, push each body to the bottom of the mold with the dorsal wall of the thorax and abdomen generally aligned along the bottom. Adjust the bodies such that the abdomen lies flat along the bottom of the mold.

Align all bodies in the X, Y, and Z dimensions to ensure each section will include all subjects simultaneously. Correct the curvature of the abdomen, especially in males, to flatten it as much as possible without puncturing or crushing the bodies. Once all subjects are in place, position the mold so that the bottom sits directly on top of a piece of dry ice.

Ensure the OCT compound begins to freeze upon contact and continue until all subjects are fully encased in the mold. After freezing, transfer the mold to a freezer set to minus 20 degrees Celsius to allow complete freezing throughout. Once fully frozen, fill the remaining portion of the mold with OCT compound.

To attach a chuck bit to the mold, apply a generous amount of OCT compound to the block. Press the bit flat onto the compound and allow it to freeze completely in the cryostat, typically within five minutes. Then release the chuck bit and OCT block from the mold.

Place the bit into the chuck, ensuring the block remains properly oriented from top to bottom and tighten the chuck key to secure the bit. Now, using the adjustment knobs and chuck depth controls, align the block with the blade and set the section width to 20 micrometers or lower. Cut each slice from the block using a slow but consistent motion, allowing the anti-roll glass to capture each slice as it is cut.

Collect the sections using warmed slides. With a razor blade, remove the excess OCT compound surrounding the sections. Outline the border of each slide using a hydrophobic marker to keep wash and stain solutions confined to the tissue.

Using a 1000-microliter pipette, wash the slides three times with PBS for five minutes each without touching the tissue. After discarding the final wash, pipette the fluorescent stain solution onto the tissues and incubate for one hour at room temperature in the dark. Then, discard the fluorescent stain and wash the slides three times for five minutes each with PBS.

Afterward, discard the final PBS wash, leaving a small amount of solution on the slide. Using a 1000-microliter pipette, pipette three to five drops of mounting media onto the slide, avoiding direct application to the tissue. Using forceps, position the cover slip at one edge and gradually lower the other side until it is flush with the slide.

Seal slides with nail polish without neon or glitter. If the mounting medium isn't hardening, depending on the fluorescent stain, image immediately or store the slides for later. Before imaging, inspect the slides for cleanliness and gently clean them using a tissue wipe with a 70%ethanol in water solution.

Analyze the brightest fluorescing subjects within the experiment to select appropriate exposure times for each fluorescence channel. Survey each experimental group in a live view for all channels to confirm correct exposure settings. For consistency, focus on each subject using the same fluorescence channel and capture images for all subjects.

After imaging, store the slides in the dark at four degrees Celsius for future image recapture. The fluorescence imaging method preserved key features of the drosophila heart, such as the ostia, valve, longitudinal fibers, and circumferential fibers. The entire heart tube structure and detailed minor structures were captured at different magnifications, with 10X for the whole structure and 40X to 60X magnifications showing finer details, such as circumferential contractile fibers and longitudinal stabilizing fibers.

Representative fluorescence micrographs demonstrated preserved heart structures in w1118 drosophila with DAPI-stained nuclei, lipids stained green, and actin-containing myofibrils stained red with phalloidin. Phalloidin staining effectively highlighted longitudinal and circumferential fibers, demonstrating the preserved actin filament structure despite freezing and sectioning stresses. Lipid spot staining revealed fat storage localized near the heart tube.