Lsm 710

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)).

Third-instar larvae were dissected, fixed in Bouin’s fixative or 4% PFA in PBS, and immunostained with previously described methods (Eaton et al., 2002 (link); Harris et al., 2015 (link)). Third instar larvae were dissected with cold HL3 and immediately fixed with PFA (4%) and incubated overnight at 4 C with primary antibodies (rabbit anti-Dlg, 1:1000; anti-Brp 1:100, Life Technologies). Alexa-conjugated secondary antibodies were used for secondary staining (Jackson Laboratories 1:500). An inverted epifluorescence deconvolution confocal microscope (Axiovert 200, Zeiss) equipped with a 100X objective (N.A. 1.4), cooled CCD camera (CoolSnap HQ, Roper Scientific) was used to acquire images. All acquisition, deconvolution and analysis were done by Slidebook 5.0 software (3I, Intelligent Imaging). Structured illumination microscopy (Nikon LSM 710 equipped with 63X objective and Andor Ixon EMCCD camera) was used to perform Brp puncta and Dlg labeling experiments. Bouton numbers were quantified as described previously (Harris et al., 2015 (link)).

AFOG and ISH images were taken using a Nikon Eclipse Ni microscope with a Nikon Digital Sight DS-Ri1 camera. Immunostaining images were taken using a Zeiss Axio Observer.Z1 microscope, Zeiss LSM 710 confocal microscope and Andor Dragonfly 500 High Speed confocal microscope.