Anti synorf1 3c11

In order to visualize the structure of the brain, immunocytochemistry with an anti-synapsin antibody was performed. The dissected brains were fixed overnight at 4 °C with a 4% paraformaldehyde solution (PFA) in phosphate-buffered saline (PBS, pH 7.4) containing 684 mM NaCl, 13 mM KCl, 50.7 mM Na₂HPO₄, 5 mM KH₂PO₄. The brains were washed in PBS four times and each for 15 min at room temperature. The brains were preincubation in 5% normal goat serum (NGS, Sigma, St. Louis, MO, USA) in PBS containing 0.5% Triton X-100 (PBST; pH 7.4) for 3 h at room temperature to block the nonspecific staining. Then, the brains were incubated with the primary antibody anti-SYNORF1 (3C11, Developmental Studies Hybridoma Bank [DSHB], University of Iowa, Iowa City, IA, USA) in PBST, 1:100 for 5 days at 4 °C. Next, the brains were rinsed six times (20 min each) in PBS at room temperature and incubated with the secondary antibody Cy2-conjugated goat anti-mouse in PBST, 3:1000 for 3 days at 4 °C. After rinsing six times (each time 20 min) in PBS, the brains were dehydrated through an ascending ethanol series (50%, 70%, 90%, 95%, 99%, 2 × 100%, 10 min each). Finally, the brains were embedded in Permount after being cleared in methyl salicylate.

Male, female and larval C. marinus were fixed in 4% paraformaldehyde (ROTH, Germany) in 0.1 M PBS overnight at 4 °C. Nervous system was prepared in 0.1 M PBS containing 0.001% Trition X (PBST). Subsequently, samples were washed six times for 30 min in 1 × PBST and thereafter blocked for 3 h in 2% normal goat serum (NGS) in 1xPBST. To label synaptic regions the monoclonal mouse anti-synapsin antibody (anti-SYNORF-1 – 3C11, Developmental Studies Hybridoma Bank, USA) was applied at a 1:500 dilution in 2% NGS-PBST and incubated for 4 days at 4 °C. Detection of the 3C11 antibody was performed by incubating in a goat-anti mouse antibody linked to Alexa Fluor 546 at a dilution of 1:200 (Invitrogen, USA) for 3 days at 4 °C. The incubation was followed by washing steps as described above. For visualization, samples were mounted in 50% glycerol on a microscope slide and scanned using confocal laser scanning microscopy (LSM 510 Meta or LSM 880, Zeiss, Germany). Alexa 546 was exited using the Helium Argon 543 laser. Signals were detected by a spectral detector (555–681 nm). All pictures were taken using a 40 × W objective (C-Apochromat 40x/1.2 W UV-VIS-NIR or C-Apochromat 40x/1.20 W Korr M27). Scanning resolution was set to 1024 × 1024 pixel. 3D reconstructions were prepared with Amira 4.1.1 (Mercury Systems, Germany).

Whole-mount immunohistochemistry (IF) was performed as in ref. ^{118 (link)}: animals were killed with cold 2% HCl and fixed with 4% FA at RT. After 4 h in blocking solution (1% BSA in PBS Triton-X 0.3%), animals were stained overnight at 4 °C. Animals were washed extensively with PBSTx, blocked for 2 h, and stained overnight at 4 °C. The following antibodies were used in these experiments: mouse anti-synapsin (anti-SYNORF1/3C11, 1:50; Developmental Studies Hybridoma Bank) and anti-Smed-β-catenin-2 (1:1000;^{119 (link)}). The secondary antibodies used were Alexa 488-conjugated goat anti-mouse (1:400; Molecular Probes; A28175) and Alexa 568-conjugated goat anti-rabbit (1:1000; Molecular Probes; A-11011). Nuclei were stained with DAPI (1:5000).