This protocol describes how to perform immunohistochemical staining on dissected gsof larvae to visualize proteins involved in synapse development. A 1.7 mil tube is used throughout the procedure for primary and secondary antibody incubation, as well as for the wash steps. Larvae are then transferred on a processing slide where they're trimmed with an exacto knife for viewing specimens are moved to a drop of anti fading reagent.
On a mounting slide, a cover slip is finally sealed to the slide with nail polish. Hi, I'm Jonathan Brent from the laboratory of Brian McCade in the Department of Physiology and Cellular biophysics at Columbia University College of Physicians and Surgeons. We use this procedure to study the genetic regulation of synapse formation at the neuromuscular junction and developing an node.
So let's get started. Before performing immunohistochemistry on the gsof larva n MJ first properly dissect the larvae. To learn this procedure, watch the accompanying video article entitled Drosophila Larva N MJ Dissection.
To begin the procedure, prepare three fresh solutions. First, make PBT Solution by adding detergent to freshly made PBS. Second, take an aliquot of bovine serum albumin and add it to a PBT solution.
To make PBTB third, take an aqua normal goat serum and add it to PB TB solution to make PBTN solution. After preparing these solutions, transfer the larvae into a 1.5 mil tube containing one mil PBT. This is the first wash.
Every wash involves replacing the solution in which the larvae are immersed and then incubating the larvae on a slowly rotating mutator mixer. Do not transfer larvae from tube to tube as this will damage them. This first wash is for 15 minutes at room temperature.
After 15 minutes of mixing, replace the PBT with fresh PBT using a pipette and wash the larvae for another 15 minutes. When exchanging wash solutions, always be careful to avoid damaging larvae When the PBT washes are complete. Wash the larvae in PBTB for 30 minutes.
Repeat this wash with fresh solution for another 30 minutes. With the PBTB wash is complete. Wash twice with PBTN for 15 minutes per wash.
During the second washing step, primary antibody can be diluted in PBTN at the desired working concentration. Following the wash, incubate the larvae in this antibody mixture for one hour with gentle mixing at room temperature or overnight at four degrees Celsius. When the incubation is complete, siphon away the primary antibody solution and quickly rinse the larvae with PBTB several times to remove any remaining antibody.
To prepare the larvae for the next antibody incubation, wash them once in PBTB for 15 minutes and wash twice with PBTN for 30 minutes. Dilute the secondary antibody in PBTN at the desired working concentration and incubate the larvae in this mixture for one and a half hours. At room temperature, make sure to cover the larvae to prevent light exposure and to minimize floor four bleaching.
After one and a half hours, remove the secondary antibody solution and rinse the larvae several more times in PBTB. Now, wash the larvae in PBTV for 15 minutes twice. All the remaining washing and storage of the larvae should be done with minimal exposure to light, so be sure to keep them covered.
Transfer the larvae from the 1.5 mil tube into PBT in a glass processing slide. The larvae are now ready for mounting to mount the stained larvae heat prolonged gold solution in a water bath at 65 degrees Celsius for two to three minutes. Then take an Eloqua and keep it on a heat block set at 50 degrees Celsius.
Apply some PB TB to a processing slide. Transfer the larvae to the PB tb. Rotate the larvae so they are cuticle side down using an exacto knife with blade number 16, trim the heads and tails from the larvae.
You can also use a razor for this step. Now prepare the mounting slide to a clean slide at 40 microliters of prolonged gold from the heated aliquot and spread it around with clean forceps. Using forceps.
Carefully transfer six to eight of the prepared larvae from the processing slide to the mounting slide. Try to keep them in the same orientation and keep their cuticle sides down to mount the larvae. Drop a cover slip over the prolonged solution.
Set it up on one edge at the periphery of the prolonged pool and carefully drop the slip to cover the samples. Seal the edges of the cover slip to the slide with your favorite color of thick, cheap nail polish. Let the slide dry for a minimum of three hours.
If the slides will be imaged only once and immediately after preparation, then the prolonged gold solution may be substituted with glycerol. Then the drying time of the nail polish and glycerol is only about 10 minutes as opposed to three hours with any staining protocol. The goal is high specific signal combined with low background.
This is achieved by using high quality antibodies and by thoroughly performing the blocking and washing steps. These images show wild type synapses as well as those from a fly strain expressing a mutated type two BMP receptor known as the wishful thinking or witch mutant. The neuronal membrane is stained blue with an antibody against horse radish peroxidase.
Synaptic vesicles shown in red are stained with antis synaptic antibody. The post synapse shown in green is stained with antibody against discs large. These results showed that mutating the type two BMP receptor wit produces a synaptic undergrowth phenotype.
This mutant was key in establishing BMPs as retrograde signals that regulate synaptic terminal buton number. We've just shown you how to perform immunohistochemistry on a esophagal larval neuromuscular junction. When doing this procedure.
It's important to execute the procedure with proper timing to ensure maximum reproducibility and low background. So that's it. Thanks for watching and good luck with your experiments.
