Anti fasii

anti-Fas II immunohistochemistry was performed as described previously [32] (link). Dissected brains were fixed in PBS with 4% formaldehyde, washed with PBS with 0.3% Triton X-100, and blocked in PBS with 5% normal goat serum and 0.3% Triton X-100. Brains were incubated 24–48 hrs with anti-Fas II (Developmental Studies Hybridoma Bank) diluted 1∶200 in blocking buffer. After washing in PBS, brains were incubated with Alexa-Fluor 488 conjugated goat anti-mouse secondary antibody (Invitrogen Molecular Probes) diluted 1∶500 in blocking buffer for 1 hour. After further washing, samples were mounted using ProLong Gold (Invitrogen). Scoring of MB phenotypes was done blind to genotype. All experiments represent n of at least 10.

Larval, pupal, and adult brains were rapidly dissected in PBS and fixed in 4% formaldehyde (Polysciences, Inc), larval for 15 min and pupal-adult for 30 min. Staining was performed as described (Kucherenko et al., 2012 (link)). Samples were mounted in 70% glycerol. The following antibodies were used: polyclonal rabbit anti-Dg 1:1000 (Deng et al., 2003 (link)), polyclonal chicken anti-GFP 1:2000 (Invitrogen), monoclonal mouse anti-Sec5 1:50 (gift from Thomas Schwarz [Langevin et al., 2005 (link)]), and anti-FasII 1:20, anti-Dlg 1:20, anti-Elav 1:20, anti-Arm 1:50, anti-FasII 1:50, anti-αPS2 1:50, and rat anti-DE-cadherin 1:50 from Developmental Studies Hybridoma Bank. Alexa 488, 568, goat, anti-rabbit, and anti-chicken 1:500 (Molecular Probes). To visualize nuclei, a 10-min-long incubation with 1× DAPI (Sigma Aldrich) in PBS was performed.

Wandering third instar larvae from sparsely populated bottles were collected and dissected in Ca2+-free saline. A detergent-free protocol was used to visualize labeling at the NMJ for anti-Sdc (1:250) [20 (link)], but all other antibodies including anti-HRP [1:2000] (Jackson Immunoresearch), anti-FasII [1:20] (Developmental Studies Hybridoma Bank), and anti-myc [1:3000] (Developmental Studies Hybridoma Bank) were used in the presence of 0.1% Triton as previously described [21 (link)]. Alexa488 goat anti-mouse, Alexa488 goat anti-rabbit, Alexa568 goat anti-rabbit and Alexa568 goat anti-mouse secondary antibodies (Invitrogen) were used at 1:500. Imaging was done on a Nikon C1 laser scanning confocal microscope. Statistical analysis of boutons per NMJ was conducted in Excel by Student’s t-test on larval pelts that were scored blind to genotype.