Processing Insect Legs for Fluorescence Microscopy: A Method to Preserve Neuromuscular Structures for Imaging
Transcrição
– To begin, submerge anesthetized insects de 70% ethanol solution para reduce the hydrophobicity of the cuticle. Then, wash and permeabilize the tissue with phosphate-buffered saline containing a detergent for at least 30 minutes para allow tissue penetration during fixation.
Next, remove the head and abdomen from the thorax. Use forceps para apply gentle pressure para the coxa-thorax junction, and detach the leg. Carefully place the legs de ice-cold 4% paraformaldehyde solution overnight para allow it para 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 para provide enough time for the buffer para enter the tissue.
Lastly, mount the legs de a drop of mounting medium. Use a spacer between the microscope slide and the coverslip para 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 de a glass multiwell plate with 70% ethanol. Use a brush para add 15 para 20 carbon dioxide anesthetized flies of any age or sex para 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 de phosphate-buffered saline for at least 10 minutes per wash. After the last wash, use forceps para 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 para gently but firmly apply pressure para the coxa-thorax junction para detach one leg from the thoracic segment.
Place the legs de 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 para push the legs gently into the fixation buffer without letting them float para obtain well-fixed legs.
– The next day, wash the legs five times de 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 de the mounting medium for at least 24 hours.
The next day, add approximately 20 microliters of 70% glycerol next para the coated end of the glass microscope slide and cover the glycerol with a 22 by 22 millimeter coverslip. Next, add de about 10-microliter line of mounting medium para the right of the coverslip and apply a second 30-microliter line of mounting medium para the right of the 10-microliter line.
Using fine forceps, transfer one leg from the multiwell plate de a drop of medium para the 10-microliter strip of mounting medium de an external side up or down orientation. Repeat until six para 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 para secure them de place.
