Dissection and antibody labeling was performed as described previously [80 (link)]. Briefly, flies were incubated in 4% PFA + 0.2% Triton-X 100 (Merck, Darmstadt, Germany) for 3.5 hours at 4°C and then washed for 3 × 30 minutes in PBS + 0.2% Triton-X 100 at room temperature prior to brain dissection. Dissected brains were incubated in primary antibodies for 2 to 5 days and in secondary antibodies for 2 days at 4°C. Afterwards, brains were washed for at least 3 × 30 minutes. The following antibodies were used in this study: rabbit anti-GFP (A6455, Thermo Fisher, Waltham, MA) (1:1,000); rabbit anti-Dlg [81 (link)] (1:30,000); rat anti-CD8a (MCD0800, Thermo Fisher, Waltham, MA) (1:500); mouse anti-Brp (nc82, Developmental Studies Hybridoma Bank, Iowa City, Iowa) (1:200); rabbit anti-dsRed (632496, Takara, Kyoto, Japan) (1:500); mouse anti-V5 (960–25, Thermo Fisher, Waltham, MA) (1:500); and Alexa488, 568, and 647 coupled secondary antibodies (Thermo Fisher, Waltham, MA) (1:1,000). Brains were mounted in Vectashield (Vector Laboratories, Burlingame, CA). Images were captured using a Zeiss LSM 700 laser scanning confocal microscope with a 25× (NA 0.8) or 40× (NA 1.3) oil immersion objective (Carl Zeiss Microscopy GmbH, Jena, Germany). Images were processed using Imaris (Bitplane AG, Imaris.oxinst.com/">www.Imaris.oxinst.com ) and Adobe Photoshop software (Adobe; www.adobe.com ).
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