Mouse anti fas2

Adult flies were fixed with 4% paraformaldehyde in PBS/0.15% Triton X-100 for 2.5 h and brains were afterwards dissected. Larval brains were dissected first and then fixed in 4% paraformaldehyde in PBS for 30 min. Brains were washed twice with PBT (0.3% Triton in PBS) before blocking in 5% normal goat serum (NGS) (5% NGS in PBT) at room temperature for 1 h, followed by incubation with primary antibodies at 4°C overnight. Primary antibodies were diluted in 5% NGS: mouse anti-TH clone LNC1 (1:200, Merck, MAB318), guinea pig anti-Lamin-DmO (1:300, kind gift from Georg Krohne, University of Würzburg, Würzburg, Germany), mouse anti-Fas2 (1:10, Developmental Studies Hybridoma Bank) or rabbit anti-GFP (1:1000, MoBiTec, A6455). Following three washing steps in PBT, brains were incubated for 4 h at room temperature with secondary antibodies: donkey anti-guinea-pig-DL650 (1:100, Thermo Fisher Scientific, SA5-10097) or donkey anti-guinea-pig-Cy3 (1:100, Dianova, 706-166-148); goat anti-rabbit-Alexa488 (1:100, Molecular Probes, A-11034); goat anti-mouse-Alexa488 (1:150, Dianova, 115-545-166), donkey anti-mouse-Cy3 (1:100, Dianova, 715-165-151) or donkey anti-mouse-Cy5 (1:100, Dianova, 715-175-151). After washing in PBT, brains were embedded in VectaShield (Vector Laboratories) and confocal images were recorded with a Leica TCS SPE microscope.

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^{10 (link)}. Polytene chromosome preparations and staining were performed as in Karachentsev et al.^{27 (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.

Embryo fixation and antibody staining were performed using standard methods. The following antibodies were used: rat anti-Pumilio (1:200, provided by Robin Wharton), mouse anti-Fas2 (1:100, Developmental Studies Hybridoma Bank), mouse anti-prospero (1:20, Developmental Studies Hybridoma Bank), rabbit anti-β-gal (1:1000, Abcam). RNA probes were made using cDNA clones obtained from the Drosophila Genome Resource Center, Indiana University, USA. Digested plasmids were used as template for in vitro transcription of DIG-labeled RNA probes (Roche Applied Science). In situ hybridization was performed using standard methods.