Secondary fluorophore conjugated antibodies

Wandering third-instar larval imaginal discs were dissected in PBS and fixed for 20 minutes in 4% PFA. Samples were rinsed in PBS. Follicles were dissected in Schneider's medium containing 15% FBS and fixed for 20 minutes in 4% PFA. Primary and secondary antibodies were diluted in PBST (0.1% Triton X-100) with 4% NGS (Gibco) and 1% BSA (Gibco). Primary antibodies (Supplemental Table 4) were incubated with samples overnight at 4°C. Secondary fluorophore-conjugated antibodies (Molecular Probes) were diluted 1:400 and incubated for 2 hours at room temperature. Phalloidin and DAPI incubated with samples for 20 minutes in PBS.
Images were captured on a Zeiss LSM700 scanning confocal microscope or a Zeiss Axio Imager M2 with Apotome 2 with Plan Apochromat 20x/NA 0.8, LD C-Apochromat 40x/NA 1.1 W and Plan Apochromat 63x/NA 1.4 oil objectives at 1024x1024 pixels with 2 line averages.
Dlg cortical enrichment was quantified as described in (Lu et al., 2021) . For single cells in en face confocal sections, the fluorescent signal intensity of at the membrane and in the cytoplasm were quantified in Fiji (Schindelin et al., 2012) using rectangular ROIs of fixed 1.17µm width, approximately the thickness of the cell cortex. The ratio of membrane:cytoplasmic intensity was calculated to give the "plasma membrane index" (PM index). Average PM indices were calculated for all cells per genotype.