Optimizing Sample Preparation Process for Transmission Electron Microscopy of Neuromuscular Junctions in Drosophila Larvae

The diffused distribution and limited visual range of the TEM present challenges for the infrastructural analysis. This protocol presents an innovative and efficient sample preparation method that surpasses the conventional approach. This advanced method of TEM sample preparation is more effective in preventing the curling of the samples compared to the traditional method by allowing the samples to stay flat during the subsequent dehydration.

The selection of reagents, and adjustment of dosage must be necessary depending on the sample type. There are many toxic chemical reagents used in the sample preparation process. Demonstrating the procedure will be Sheng Qingyuan, a master student from Dr.Gan Guangming's laboratory.

To begin, dissect the wandering late third instar Drosophila larvae in Jan solution, and obtain the entire body wall per the standard procedures. Fix the larval body wall with the fixative in the dissection chamber overnight at four degrees Celsius. The next day, transfer the larval body wall from the dissection chamber to a 1.5 milliliter centrifuge tube, and rinse it several times with sodium cacodylate buffer.

Fix the larval body wall in 1%osmium tetroxide for two hours. After fixation, wash the samples several times with distilled water. Add 2%saturated urinal acetate solution into the tube to stain the PHI neuromuscular junction.

And after two hours, rinse the samples with deionized water. Next using scissors, cut two round pieces of metal mesh of pore size 270 microns. Place the metal mesh at the base of a flat bottomed bottle.

Then place the fixed larvae on the mesh, followed by placing another mesh. Dehydrate the samples in an increasing ethanol concentration for 20 minutes each at four degrees Celsius. Then rinse the samples twice with propylene oxide at room temperature for five minutes each.

Next, place the samples in a propylene oxide and epoxy resin solution, followed by pure epoxy resin for two hours at room temperature. For embedding, cut a polyethylene film into a big square and a small, hollow rectangular support. Glue the small square with the big square.

Add sufficient epoxy resin at the center of the big square. Then turn on the stereoscope, and place the samples in the pre-positioned epoxy resin with the muscle side facing upwards. Add several drops of epoxy resin onto the samples, and cover them with polyethylene film.

Place the samples for polymerization at increasing temperatures for 24 hours each. After polymerization, using a sharp blade, remove the excess parts of the larval body while retaining a trapezoid, including the A2 and A3 segment. Remove the trapezoid from the remaining larval body wall, and attach it to the resin-filled capsule with AB glue.

Under a light microscope, confirm the position of the sixth and seventh muscles of the A2 and A3 segments, maintaining the tangent plane parallel to the sixth muscle. Then cut away one-fifth of the sample along the two sides of the A3 segment. Using a microtome, prepare an ultra thin section of the sample starting from the sixth muscle.

After sectioning, attach as many slices as possible to each copper mesh. Use a transmission electron microscope to observe the samples. Among Lowicryl K4M resin and epoxy resin, Lowicryl K4M resin provided sharper and more easily observable results of the Drosophila body muscles.

Confocal imaging demonstrated enhanced visualization of the neuromuscular junction between the sixth and seventh muscles. Transmission electron microscopy revealed bead-like neuromuscular junctions, offering improved synaptic structure information. The samples remained flat throughout the dehydration process due to the restriction of the metal meshes.

Besides, the samples were polymerized between the plastic products, preventing the samples from folding. In the future, the TEM sample preparation method can be applied to brain and ventral nerve cord neuropil, thereby improving the potential of ultrastructural research.