Third-instar larvae were dissected, fixed in Bouin’s fixative or 4% PFA in PBS, and immunostained as previously described (Eaton et al., 2002 (link); Harris et al., 2015 (link)). Dissected third instar larvae were fixed with PFA (4%) and incubated overnight at 4 C with primary antibodies (mouse anti Flag 1:50; rabbit anti-RFP 1:100; rabbit anti-Dlg, 1:1000; anti-Syt1 1:1000, anti-Brp 1:100, Life Technologies). Alexa-conjugated secondary antibodies and goat anti-HRP were used (Jackson Laboratories 1:500). Images were acquired with either a Zeiss LSM700 confocal microscope equipped with Zen software using a 63X 1.6 NA oil immersion objective or an upright epifluorescence deconvolution confocal microscope (Axiovert 200, Zeiss) equipped with a 100X objective (N.A. 1.4), cooled CCD camera (CoolSnap HQ, Roper Scientific). Slidebook 5.0 (3I, Intelligent Imaging) was used for capturing, deconvolving and analyzing images. Structured illumination microscopy (Nikon LSM 710 equipped with 63X objective and Andor Ixon EMCCD camera) was used to perform Brp-GFP and MCTP-Flag colocalization experiments. Bouton numbers and Brp numbers and densities were quantified as described previously (Harris et al., 2015 (link)).
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