Cyanine 3 conjugated goat anti rabbit

Dechorionated, formaldehyde-fixed, methanol devitellinized embryos were fluorescently stained as previously described (Bashaw, 2010b ). Live-dissected embryos were stained as previously described (Bashaw, 2010a ). The following primary antibodies were used: mouse anti-1D4/FasII [Developmental Studies Hybridoma Bank (DSHB); 1:100], mouse anti-Beta gal [DSHB; 1:150], mouse anti-Robo [DSHB; 1:50], mouse anti-Myc [DSHB (9E10); 1:500] rabbit anti-GFP [Invitrogen #A11122); 1:500], mouse mAb anti-V5 [Serotec; 1:200], Mouse anti-HA [Covance (16B12) 1:250], Mouse anti-Sema2a [DSHB 1:10], Alexa647-conjugated goat anti-HRP [1:500, Jackson Immunoresearch (#123-605-021); 1:500]. Cyanine 3-conjugated goat anti-rabbit [Jackson; 1:1000], Alexa488-conjugated goat anti-mouse [Molecular Probes; 1:500] were used as secondary antibodies. Images were acquired using a spinning disk confocal system (PerkinElmer) built on a Nikon Ti-U inverted microscope using a Nikon OFN25 60X or 40X objective with a Hamamatsu C10600-10B CCD camera and Yokogawa CSU-10 scanner head with Volocity imaging software. Images were processed using ImageJ.