The overall goal of this procedure is to conduct an immunofluorescent stain for active zone proteins in drosophila third and star larvae. This is accomplished by first selecting and pinning the larvae down onto the dissection dish. The second step of the procedure is to dissect the larvae, removing excess tissue to expose the muscles and neuromuscular junctions.
The third step of the procedure is to immunostain the dissected larval pelts directly in the dissection dish. The final step of the procedure is to mount the larval pelts on a microscope slide. Ultimately, results can be obtained the CHO neuromuscular junction active zone morphology through immunofluorescence microscopy.
This method can help answer key questions in the neurology field, such as regulation of neuromuscular junction proteins like once involved in active zone morphology To create a dissecting surface, pour cigar, 180 4 silicone elastomer base into a small tissue culture plate. Make sure not to fill the plate completely so that the dissection area is lower than the rim Trim dissection pins to a length of approximately three to five millimeters for increased ease of manipulation, and make sure to have at least six pins per larvae from wild type and mutant strains. Identify and select wandering third instar larvae that are actively crawling around the wall of the vial and have not begun to pupate.
Remove two larvae from each genotype from the vials and place them in a washing dish filled with PBS to remove debris under a stereo microscope. W away excess moisture on the dissecting surface Using a kim wipe. Arrange the larvae so that the dorsal side is facing up and the two white trachea are visible along the dorsal side of the larvae Center.
The tracheas in the middle of the larvae as they mark its dorsal midline place. PIN number one just above the mouth hooks, and then place pin number two in the tail position just above the sphericals and in between the tracheas. Add a small amount of PBS to the larvae to prevent drying of the cuticle.
Be sure to keep different genotypes separated by placing a pin between each group. Once the larvae have been pinned down, make a transverse cut across the tracheas just above pin number two. Insert your scissors in this cut and cut up along the dorsal midline between the tracheas all the way up past pin number one if needed.
Make four additional small cuts at the very top and bottom of each side of the cuticle so that the left and right sides will open up, allowing you to pin them down without pulling on the cuticle. Once each corner of the cuticle is pinned down flat against the dissecting pad, fix the filet larvae with 4%para formaldehyde for 20 to 25 minutes at room temperature. After about 25 minutes of fixation time, decant the paraform aldehyde and rinse with PB S3 times decanting each time with the larvae in a drop of PBS under the stereo microscope, grab the posterior end of one of the trachea with dissecting forceps and pull it out.
Repeat for the second trachea. Grab some of the posterior fat body or intestine tissue and pull it out of the dissecting area. Once most of the posterior tissue has been removed, focus on removing the small tissues around the brain.
Once the larval pelt is free of excess tissue, it can be stained directly on the dissecting pad using a primary antibody to recognize the active zone, followed by a fluorescent secondary antibody After immunofluorescent staining of the active zone, the next step is to mount the immunostain larval pelts. Begin by decanting and wicking off the final wash following secondary antibody incubation and pipette approximately one milliliter of 80%glycerol. To cover the pin down larva pelts, remove all but one pin from each larvae and then place two drops of mounting media onto a microscope slide.
Remove the final pin and grab the larvae by the tail with blunt forceps. Drag the larvae around in the glycerol and then place the larvae in the first mounting medium drop and do the same with remaining larvae of the same genotype. Once all the larvae are in the first mounting medium drop, grab the larvae one by one and drag them through the drop and then along the dry edges of the slide to remove a majority of the medium.
Then place the larvae in the second mounting medium drop. Prepare the final slide by labeling it and placing a small T shape of mounting medium on the slide. Repeat the drag and dry procedure with the larvae in the second mounting medium drop, and then arrange the larvae cuticle side down in the mounting medium.
On the final slide, place a cover slip on the slide to cover the dissected larvae with the starting edge In the upper line of the tee place a small weight on the cover slip for about five minutes to ensure that the mounting media spreads out completely, and then let it sit in the dark until the mounting medium has cured. To prepare the slide for microscopy, wipe off any residual mounting media and seal the cover slip with fingernail polish. The proper orientation of mounted larval pelts is shown here.
Notice that the pelts are flat cuticle side down and that there is at least a larval body width in between pelts. Once the larval dissections, staining and mounting are finished. Microscopy can be used to identify the larvae muscle of interest located in segments a two through a six.
Using a high power objective, one can focus on a single synapse and take a ZS stack image with a microscope. Shown here is a representative maximum projection image of a portion of synapse with NC 82 staining in green and HRP pre-synaptic stain in red. After watching this video, you should have a good understanding of how to immunostain for important neuromuscular junction proteins like ones involved in active zone morphology.
