Processing Insect Legs for Fluorescence Microscopy: A Method to Preserve Neuromuscular Structures for Imaging

- To begin, submerge anesthetized insects in 70% ethanol solution to reduce the hydrophobicity of the cuticle. Then, wash and permeabilize the tissue with phosphate-buffered saline containing a detergent for at least 30 minutes to allow tissue penetration during fixation.

Next, remove the head and abdomen from the thorax. Use forceps to apply gentle pressure to the coxa-thorax junction, and detach the leg. Carefully place the legs in ice-cold 4% paraformaldehyde solution overnight to allow it to penetrate the tissue. Paraformaldehyde fixes tissues by cross-linking proteins.

Then, remove the fixation solution and wash the tissue several times with phosphate-buffered saline containing detergent. Before mounting, place the legs in pure or slightly diluted mounting buffer for at least one day to provide enough time for the buffer to enter the tissue.

Lastly, mount the legs in a drop of mounting medium. Use a spacer between the microscope slide and the coverslip to prevent the specimen from damage, and secure the mount with nail polish.

In the following example, we will see the dissection, fixation, and mounting of Drosophila melanogaster legs.

- Begin by filling the appropriate number of wells in a glass multiwell plate with 70% ethanol. Use a brush to add 15 to 20 carbon dioxide anesthetized flies of any age or sex to each well, and gently dab the flies into the ethanol until they are fully submerged.

After no more than one minute, rinse the flies three times with 0.3% nonionic surfactant detergent solution in phosphate-buffered saline for at least 10 minutes per wash. After the last wash, use forceps to remove the head and abdomen of each fly without damaging the thoracic segment or the legs, and use the tip of a pair of fine forceps to gently but firmly apply pressure to the coxa-thorax junction to detach one leg from the thoracic segment.

Place the legs in one well of a new multiwell plate, containing freshly prepared 4% paraformaldehyde on ice, for an overnight incubation at 4 degrees Celsius.

- It is important to push the legs gently into the fixation buffer without letting them float to obtain well-fixed legs.

- The next day, wash the legs five times in fresh 0.3% nonionic surfactant detergent solution for 20 minutes per wash. After the last wash, replace the detergent with mounting medium and keep the legs in the mounting medium for at least 24 hours.

The next day, add approximately 20 microliters of 70% glycerol next to the coated end of the glass microscope slide and cover the glycerol with a 22 by 22 millimeter coverslip. Next, add in about 10-microliter line of mounting medium to the right of the coverslip and apply a second 30-microliter line of mounting medium to the right of the 10-microliter line.

Using fine forceps, transfer one leg from the multiwell plate in a drop of medium to the 10-microliter strip of mounting medium in an external side up or down orientation. Repeat until six to eight legs have been mounted and aligned, and place a second coverslip over the legs such that the second coverslip rests slightly on the first coverslip. Then, use nail polish at each corner of the coverslips to secure them in place.