Immunolabeling and Imaging of Drosophila Embryos

The following antibodies were used for immunolabelling of late stage embryos and chromosomal preparations: mouse anti-Fas2 (Developmental Studies Hybridoma Bank, DSHB), rabbit anti-HRP (Jackson Immunoresearch), mouse anti-Connectin (DSHB), rabbit anti-dsRED (Invitrogen), rabbit and mouse anti-GFP (Invitrogen), mouse anti-H3K4me3 (Invitrogen). Secondary antibodies were purchased from Invitrogen. DNA was labeled with DAPI (Invitrogen). Embryos were collected and fixed via a formaldehyde/MeOH method [13 (link)]. Polytene chromosome preparations and staining were performed as in Karachentsev et al. [21 (link)]. Images of the ventral nerve cord were obtained using a Leica SP8 using a 40x Objective. Fas2 and HRP labeled embryos were imaged and typically contained 8–10 hemisegments. Hemisegments were examined for midline crossing and in some instances the presence or integrity of the most lateral Fas2+ longitudinal track. Similar imaging and analysis were performed on Connectin/HRP labeled embryos.