Duolink mouse rabbit in situ pla kit

Brains were processed as described and stained using rabbit anti-GFP antibodies at 1:500 (ThermoFisher Scientific, Catalog #A-11122, Waltham, MA) with FITC-conjugated secondary antibodies and mouse anti-HA antibodies at 1:250 (Sigma-Aldrich, Catalog #A2095, St. Louis, MO) with Alexa647-conjugated secondary antibodies, leaving the red channel open. For PLA, we used the DuoLink Mouse Rabbit in situ PLA kit (Sigma-Aldrich, Catalog #DUO92101, St. Louis, MO). Following the last wash after secondary antibody incubation, the brains were incubated in the anti-mouse and / or anti-rabbit PLA probes at a 1:5 dilution for 2 hr at 37°C. Brains were then washed thrice for 10’ each with Wash Buffer A, and incubated in Ligation solution (1:40 ligase in ligation buffer) for 1 hr at 37°C. Brains were washed in Wash buffer A for three times at 10’ each and then incubated in Amplification solution (1:80 dilution of polymerase in Amplification buffer) for 2 hr at 37°C. Finally, brains were washed three times for 10’ each in Wash Buffer B, and incubated in SlowFade overnight before mounting. Controls without Probes went through the identical process as those with probes, but with water substituted for the probes themselves in the first PLA step. Brains were imaged as described via confocal microscopy.

Third instar larvae were dissected in cold HL3 solution^{62 (link)} and were incubated with anti-BRP (1:250, DSHB, Iowa city, IA)^{55 (link)} and anti-Par-1 antibodies (1:10,000, gift from Bingwei Lu, Stanford School of Medicine, CA) overnight at 4 °C. Cy5-conjugated anti-HRP antibody raised in Goat was used (Jackson ImmunoResearch) at 1:500 to label the neuronal membranes. For PLA, Duolink Mouse Rabbit in situ PLA kit (Sigma-Aldrich, St. Louis, MO) was used and the PLA assay was performed as previously described^{29 (link),63} . Synaptic boutons and axon bundles passing over A3–4 were imaged using Nikon C1 confocal microscope and analyzed as described above. At least 4 larvae from each time point and 10 NMJs were analyzed. Analysis of average PLA signal intensity was performed using MetaMorph software (Molecular Devices, Sunnyvale, CA, USA) as described in the confocal microscopy analysis section (above) and normalized to HRP intensity.