For quantification of substrate penetration into fly brains, samples from GH146-Gal4 > myrSNAP or GH146-Gal4 > myrHalo or brp-SNAP animals were incubated with 1 µM BG-549 or 1 µM Halo-TMR for 15 min, 30 min, 1 h, 4 h or 12 h. Samples from GH146-Gal4 > myrGFP or Canton-S animals were incubated with anti-GFP (1:1,000) or anti-nc82 (1:30) antibodies for 15 min, 30 min, 1 h, 4 h or 12 h, followed by washing and incubation with fluorescent secondary antibodies (anti-chicken Alexa 568 and anti-mouse Alexa 568, respectively) for either 2 days or for the same time as primary antibodies (i.e. 15 min, 30 min, 1 h, 4 h or 12 h).

Quantification of GH146 labeling

All 3D confocal stacks were registered onto a GH146-Gal4 > myrGFP template. GH146-positive areas were segmented from each registered brain using a binary mask (surfaced rendered in [fig:S_analysis]B) constructed as follows: five GH146-Gal4 > myrGFP brains (stained with anti-GFP antibody for 12 h) were averaged in ImageJ. The resulting image stack was filtered (median + Gaussian) and thresholded.

Quantification of Brp-SNAP/nc82 labeling

All images were registered against an nc82 template brain (IS2[#Cachero:2010p2371]). Two binary masks were made in order to separate neuropil and cortex (see [fig:S_analysis]A): five brp-SNAP brains labeled with BG-549 were averaged, filtered (median + Gaussian) and thresholded. In order to assess cortical background labeling, signal was quantified in the region resulting from subtraction of the neuropil mask from the whole brain mask (see [fig:S_analysis]A). For the high-resolution 3-color labeling analysis of PN terminals in the MB calyx (Figure 3E), raw image stacks were deconvolved using Huygens Professional Deconvolution software (VSI, Netherlands). Further co-localization analysis of Brp puncta was performed with the same software package and montaging was performed on deconvolved stacks in ImageJ.