Rat anti mcd8a

Fly brains were dissected in 1X PBS and fixed for 20 min at room temperature in 1X PBS containing 4% formaldehyde (Sigma: F8775). Fixed brains were washed 3 X 20 min in 1X PBS with 0.5% Triton X-100 (PBST; Sigma: T8787), followed by incubation in PBST with 5% normal goat serum (blocking solution; Jackson ImmunoResearch: 005-000-121) for 30 min. Brains were then incubated overnight with primary antibodies in blocking solution at 4°C, washed 3 X 20 min in PBST, before incubating overnight with secondary antibodies in PBST at 4°C. Brains were then washed 3 X 20 min in PBST and mounted on glass slides in Gold anti-fade reagent (Thermo Fisher Scientific: S36937). Antibodies used in this study are Mouse anti-Brp (1:50; DSHB: nc82), Rat anti-mCD8a (1:100; Thermo Fisher Scientific: MCD0800, lot# 1968949), chicken anti-GFP (1:5000; Abcam: 13970, lot# GR23665112), rabbit anti-Dsred (1:500; Clontech: 632496, lot# 1509043), donkey anti-chicken-488 (1:400; Jackson ImmunoResearch: 131753, lot# 131753), Goat anti-rat-488 (1:400; Thermo Fisher Scientific: A11006, lot# 1728142) and Goat anti-Rabbit-Cy3 (1:400; Jakson ImmumoResearch: 111-165-144, lot# 123834). Brains were imaged using an LSM 880 confocal microscope (Zeiss) and images were analyzed in Fiji/ImageJ^{61 (link)}.