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

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

Next, remove the head and abdomen from the thorax. Use forceps に apply gentle pressure に the coxa-thorax junction, and detach the leg. Carefully place the legs で ice-cold 4% paraformaldehyde solution overnight に allow it に 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 に provide enough time for the buffer に enter the tissue.

Lastly, mount the legs で a drop of mounting medium. Use a spacer between the microscope slide and the coverslip に 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 で a glass multiwell plate with 70% ethanol. Use a brush に add 15 に 20 carbon dioxide anesthetized flies of any age or sex に 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 で phosphate-buffered saline for at least 10 minutes per wash. After the last wash, use forceps に 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 に gently but firmly apply pressure に the coxa-thorax junction に detach one leg from the thoracic segment.

Place the legs で 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 に push the legs gently into the fixation buffer without letting them float に obtain well-fixed legs.

– The next day, wash the legs five times で 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 で the mounting medium for at least 24 hours.

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

Using fine forceps, transfer one leg from the multiwell plate で a drop of medium に the 10-microliter strip of mounting medium で an external side up or down orientation. Repeat until six に 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 に secure them で place.