A protocol provides a straightforward model for assessing synaptic integrity in an age dependent manner. So we can study neurodegenerative diseases in tissues that are amenable to both structural and functional analysis. This technique facilitates the preservation of both neuronal and muscle tissue from the dorsal longitudinal muscle neuromuscular junctions.
To comprehensively investigate synaptic function over time and tissues that are difficult to work with. This method can be applied to the study of neurodegenerative diseases including ALS, Parkinson's, Huntington's and Alzheimer's disease. Before beginning the dissection cut approximately 10 millimeters from the tip of a 200 microliter pipette.
After administering anesthesia, play six to 10 flies in a six centimeter Petri dish containing 70%ethanol and use a paint brush to gently sip merge each fly. After one to two minutes, use forceps to transfer one fly by the legs or wings to a silicone elastomer coated dissection dish containing seven to 10 milliliters of PBS under a dissecting microscope. With the fly submerged in the PBS, use blunt Dumont number five find forceps to carefully remove the wings and use span a straight edge spring dissection scissors to make a small incision in the ventral side of the cuticle to remove the legs.
Holding the dissection scissors in one hand and blunt forceps in the other, position the fly ventral side up and holding the specimen in place with the forceps, remove the head and abdomen with the scissors. Use a 200 microliter pipette set to 40 microliters and the modified pipette tip to collect the thoraces and place the isolated tissue samples into an appropriately labeled tube containing 900 microliters of PBS and 150 microliters of 32%formaldehyde. After 30 minutes at room temperature, use a Pasteur pipette to remove the fixative and rinse the plies three times with 1.5 milliliters of PBS per wash.
Before beginning the bisections Don cryo protective gloves and safety glasses and fill a tour flask with liquid nitrogen. Use a blade breaker to grab a feather blade at an angle and bend the blade to break off a small piece. Use the blade breaker to lock the blade in position and use a glass Pasteur pipette to remove all of the PBS from the sample tubes.
Use cryogenic tweezers to submerge the tubes in the flask of liquid nitrogen. After 10 seconds, use a Pasteur pipette to add approximately 300 microliters of ice cold PBS to each tube on ice, and place one poor Axe ventral side up in a 10 centimeter silicone elastomer coated dissection dish containing ice cold PBS. Use a dull pair of forceps to position the thorax and to find pair of forceps, to remove some of the thoracic ganglia to expose the midline of the thorax.
Using the midline of the thorax as a guide, use the play to make a shallow cut through one third of the thorax and use the blunt forceps to position the thorax at a 45 degree angle. Use the blade to cut straight down the midline of the thorax to obtain two hemithoraces and to carefully make one or two cuts to remove the excess tissue without damaging the dorsal longitudinal muscles. Then place the muscle tissue samples into an appropriately labeled tube of PBS.
When all of the thorax samples have been bisected permeabilize the tissues in blocking buffer for at least one hour at four degrees Celsius. At the end of the incubation, vortex freshly prepared structural stain solution and add 150 microliters of the stain to each sample for a two hour incubation on a rotator at room temperature in the dark. After staining wash the samples in PBST, and place five reinforcement labels stacked and cut in half 15 millimeters apart on one glass microscope slide.
Use a modified micro pipette tip to transfer the Thoraces onto each slide and use a laboratory wipe to remove any excess PBST. Use forceps to arrange the samples such that the Thoraces are facing muscle side up. When the samples are correctly oriented use a 200 microliter pipette tip to apply 70 microliters of mounting medium to each slide and cover each slide with a cover slip.
Then use nail Polish to generously coat the outside edges of the cover slips to obtain a complete seal around each tissue. To assess the synaptic integrity. Neuromuscular junctions can be stained with horseradish peroxidase and Floe tin Motor neurons in tar binding protein of 43 Kilodalton mutans have little to no horseradish peroxidase staining by day 21, while the wild type protein remains intact.
No visible differences in muscle staining are observed. In addition to structural staining dorsal longitudinal muscle neuromuscular junction labeling can also provide an assessment of synaptic integrity with pre synaptic and post synaptic markers. Note that liquid nitrogen flash freezing facilitates a successful bisection as non flash frozen tissue is more susceptible to damage during the dissection.
Following this procedure the preserve samples can be analyzed by confocal microscopy to measure a specific aspects of synaptic structure.
