Cy5 goat anti mouse

Rabbit polyclonal anti-SERT antibody (PC177L) and mouse monoclonal anti-synaptophysin antibody (MAB5258) were purchased from Merck Millipore (Bayswater, Australia). Mouse monoclonal anti-PSD95 antibody (6G6-1C9) and rabbit polyclonal anti-gephyrin (Ab32206) antibody were purchased from Abcam (Melbourne, Australia). Goat anti-rabbit-Alexa488, goat anti-rabbit Alexa555, goat anti-mouse-Cy5, and goat anti-mouse-Alexa405 antibodies were purchased from Life Technologies (Mulgrave, Australia). Normal mouse (NMS), normal rabbit (NRS), normal goat (NGS) sera, goat F(ab) anti-mouse and anti-rabbit antibodies were purchased from Abcam (Melbourne, Australia) (Table 1).Table 1

Antibodies used in this study

Antigen	Host	Immunogen	Supplier	Catalog #	Dilution	References
SERT	Rabbit	Synthetic peptide corresponding to amino acids 602–622 of rat 5-HT transporter	Millipore	PC177L	1/1000	Zhou et al. (1996 (link))
Synaptophysin	Mouse	Vesicular fraction of bovine brain	Millipore	MAB5258	1/500	Tabuchi et al. (2007 (link))
PSD95	Mouse	Purified recombinant rat PSD-95	Abcam	Ab2723	1/1000	Tomer et al. (2014 (link))
Gephyrin	Rabbit	Synthetic peptide conjugated to KLH derived from within residues 700 to the C-terminus of Mouse Gephyrin	Abcam	Ab32206	1/1000	Nunez-Para et al. (2013 (link))

For whole mount immunohistochemistry, fly brains were fixed in 4% paraformaldehyde (PFA) for 30 min, dissected, and blocked in 5% normal goat serum for 1 hr at RT. Primary and secondary antibodies were incubated at 4°C overnight. The following primary antibodies were used: rabbit anti-GFP (Molecular Probes, Eugene, OR, Cat# A-21312, RRID:AB_221478) at 1:500; mouse anti-GFP (Thermo Fisher Scientific, Waltham, MA, RRID:AB_221568) at 1:500; rabbit anti-RFP (Rockland Cat, Limerick, PA, # 600-401-379, RRID:AB_2209751) at 1:500; and mouse anti-BRP (DSHB, Iowa City, IA, Cat# nc82, RRID:AB_2314866) at 1:150. The secondary antibodies, Alexa Fluor 488 goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA, Cat# A11008, RRID:AB143165), Alexa Fluor 568 goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA, Cat# A11011, RRID:AB_143157), and Cy5 goat anti-mouse (Thermo Fisher Scientific, Waltham, MA, Cat# A10524, RRID:AB_2534033) were used at 1:1000. Images were obtained on a Leica SP8 confocal microscope.

For whole mount immunostaining, fly brains were fixed in 4% paraformaldehyde (PFA) for 30 min, dissected, and blocked in 5% normal goat serum for 1 hr at RT. The following primary antibodies were used: rabbit anti-GFP (Molecular Probes, Eugene, OR, Cat# A-21312, RRID:AB_221478) at 1:500, mouse anti-RFP (Rockland Cat, Limerick, PA, # 600-401-379, RRID:AB_2209751) at 1:500, BRP (DSHB, Iowa City, IA, Cat# nc82, RRID:AB_528108) at 1:150; anti-HA (Covance Research Products Inc, Princeton, NJ, Cat# MMS-101R-500, RRID:AB_10063630) at 1:1000; and anti-DSX (kind gift from Bruce Baker) at 1:300. The secondary antibodies, Alexa Fluor 488 goat anti-rabbit (Thermo Fisher Scientific, Waltham, MA, Cat# A11008, RRID:AB_143165) and Cy5 goat anti-mouse (Thermo Fisher Scientific, Waltham, MA, Cat# A10524, RRID:AB_2534033) were used at 1:1000. Primary and secondary antibodies were incubated at 4°C overnight. For GRASP experiments, fly brains were fixed in PFA for 30 min at RT, and imaged without immunostaining. Images were obtained on a Leica SP8 confocal microscope.

Zebrafish samples were prepared for immunofluorescence as previously described (Fausett and Goldman, 2006 (link); Ramachandran et al., 2010a (link); Ramachandran et al., 2010a (link)). Primary antibodies used in this study: anti-pSmad3, Abcam Cat. # ab52903 (1/200); Zpr-1 and Zn-5, Zebrafish International Resource Center (1/500 and 1/1000, respectively); anti-HuC/D, Abcam, Cat. #ab210554 (1/500); anti-PKC_β1, Santa Cruz Biotechnology, Cat. #SC-8049 (1/200); anti-glutamine synthetase (GS), Sigma-Aldrich, Cat. #MAB302 (1/500); anti-SOX9, EMD Millipore, Cat. #AB5535 (1/500); anti-BrdU, Thermo Fisher, Cat. # MA 1–82088 (1/500) and Cat. # B35128 (1/500, clone MoBu-1 for co-staining with EdU Click-It chemistry). Secondary antibodies: Alexa Flour 555 Donkey anti Mouse-IgG (H+L), Thermo Fisher Cat. # A31570 (1:500); Alexa flour 555 Donkey anti Rabbit IgG (H+L), Thermo Fisher, Cat # A31572 (1:500); Alexa flour 555 Donkey anti Sheep IgG (H+L) Thermo Fisher Cat #A21436. Cy3, Jakson Immuno research labs, Cat #712-166-150 (1:500); Alexa Flour 488 donkey anti mouse Thermo Fisher Cat. # A21202 (1:500); Alexa Flour 488 goat anti rabbit Thermo Fisher Cat. # A11008 (1:500); Cy5 goat anti mouse, Thermo Fisher Cat. # A10524 (1:500); and Alexa Flour 647 goat anti rabbit Thermo Fisher Cat # A21244 (1:500). In situ hybridization was performed as described previously (Barthel and Raymond, 2000 (link)).