Ss8x9 secureseal spacers

Immunostaining of whole-mount brains or VNCs was performed as follows: brains or VNCs of 3- to 7-day-old flies were dissected in PBS, fixed in 4% paraformaldehyde (PFA) for 20 min, and subjected to chicken anti-GFP (1:1000, In-vitrogen), rabbit anti-TH (1:1000, Millipore), rat anti-CD8 (1:200, Invitrogen), mouse anti-nc82 (1:25, Developmental Studies Hybridoma Bank), or all four at 4°C for 18–40 hr, followed by incubation with fluorescent Alexa 488 anti-chicken (1:1000, Invitrogen), Alexa 488 anti-rat (1:200, Invitrogen), Alexa 568 anti-rabbit (1:1000, Invitrogen), Alexa 647 anti-mouse (1:1000, Invitrogen), or Cy3 anti-mouse (1:200, Jackson Immunoresearch) secondary antibodies for ~16 hr at 4°C. Brains or VNCs were mounted in Vectashield (Vector Laboratories) or SlowFade Gold using SS8X9-SecureSeal spacers (both Thermo Fisher Scientific). Images were obtained on an LSM510 or LSM700 confocal microscope (Zeiss) with 1-μm- or 3-μm-thick sections under 10x or 25x magnification. MultiColor FlpOut (MCFO) analysis of select driver combinations was performed as described previously (Nern et al., 2015 (link)) using rat anti-FLAG (1:200, Novus Biologicals) and rabbit anti-HA (1:300, Cell Signaling Technology) antibodies.