Anti β gal

Transfected neurons were fixed at 3 days post-transfection. Immunocytochemistry was performed as described previously^{11 (link),51 (link)}. First, hippocampal neurons were fixed in 2% formaldehyde/1 × PBS/4% sucrose for 10 min followed by incubation in cold methanol (−20 °C) for 10 min. Fixed neurons were incubated with primary antibodies diluted in 1 × GDB (0.1% gelatin, 0.3% Triton X-100, 0.45 M NaCl, 17.7 mM sodium phosphate buffer, pH 7.4) in a humidified container overnight at 4 °C. Antibodies used and their dilution factors are: anti-GABA_AR γ2 (1:250; Synaptic Systems), anti-GABA_AR α2 (1:250; Synaptic Systems), anti-vGAT (1:800; Synaptic Systems), anti-NL2 (1:500; Synaptic Systems), anti-gephyrin (1:1000; Synaptic Systems), anti-β-Gal (Promega), and anti-myc (1:100; Santa Cruz Biotechnology).
Alexa 488, Cy3- or Cy5 conjugated secondary antibodies were used to visualize bound primary antibodies.

Flies were dissected in Grace's media and ovaries were fixed and stained as described previously (Tanner et al., 2011 (link)). Samples were mounted in VectaShield with DAPI (Vector Labs). Primary antibodies used were: anti-cleaved-Dcp-1 (1:100, Cell Signaling Technology), anti-Dlg [1:100, Developmental Studies Hybridoma Bank (DSHB)], anti-αPS3 (1:300, Shigeo Hayashi, or 1:1000), anti-βPS (1:10, DSHB), anti-Drpr (1:50, DSHB 5D14), anti-β-Gal (1:400, Promega), anti-aPKC (1:1000, Santa Cruz Biotechnology, Inc.), anti-Dynein (1:3, DSHB 2C11), anti-Kinesin (1:100, Cytoskeleton, Inc.), anti-Crumbs (1:25, DSHB Cq4, protocol from Tanentzapf et al., 2000 (link)) and anti-Talin (mixture of A22A and E16B, 1:50 each, DSHB). The anti-αPS3 antibody was made using the peptide sequence utilized for the original antibody (Wada et al., 2007 (link)) and generated by YenZym (San Francisco, CA). The serum was affinity-purified twice before use. It shows the same expression pattern as the original antibody from the Hayashi lab. Secondary antibodies used were goat-anti-rabbit Cy3, goat-anti-mouse Cy3, goat-anti-mouse Alexa Fluor 647 (Jackson ImmunoResearch), each at 1:100, and goat-anti-rabbit Alexa Fluor 488 (Invitrogen) at 1:200. Egg chambers were imaged on an Olympus FV10i confocal microscope, images were processed using ImageJ and Adobe Photoshop, and figures were made using Adobe Illustrator.