Rabbit anti synapsin

Immunohistochemistry was performed as previously described20 (link). Antibodies used were: mouse monoclonal anti-GFP 1:250 (Millipore), rabbit anti-synapsin 1:1000 (Synaptic Systems)22 (link), rabbit anti-PSD95 (Abcam)23 (link), Cy-3 anti-rabbit 1:400, Alexa 488 donkey anti-mouse 1:400 (Invitrogen), and rabbit anti-goat Alexa 555 (Invitrogen). Double immunohistochemistry for GFP with anti-synapsin or anti-PSD95 was performed at 3 dpf as follows: embryos were fixed in 4% paraformaldehyde (PFA) in PBS for 1.5 hours at room temperature (RT) and washed briefly in PBS (3 × 5 min) with 0.1% Triton X-100 (PBST). Embryos were then blocked (PBS with 1% BSA, 1% DMSO, 2% goat serum, and 0.1% Triton X-100) for 3 hours at RT, and then incubated in blocking buffer with primary antibodies overnight at 4 °C. Embryos were washed with PBST, and incubated with secondary antibodies overnight at 4 °C.
For sectioning, stained larvae was cryoprotected in 30% of sucrose with PBS for at least 2 hr, rinsed briefly in Tissue-Tek O.C.T. compound (Sakura Finetek, Torrance, CA), and frozen in a Tissue-Tek O.C. ethanol/dry ice bath. Sections were 20 μm and mounted on the Superfrost Plus Microscope slides.

The following primary antibodies were used: rabbit polyclonal anti-human NSun2 (1:1,000; Meta) (Frye & Watt, 2006 (link)), rabbit polyclonal CUK-1079-A anti-mouse NSun2 (1:1,000; Covalab) (Blanco et al, 2011 (link)), mouse monoclonal anti-NMP1 (1:500; Sigma, B0556, clone FC82291), mouse monoclonal anti-angiogenin (1:200; Abcam, ab10600, clone 14017.7), goat polyclonal anti-eIF4AI (1:200; N-19) (Santa Cruz, sc-14211), goat polyclonal anti-4ET (1:200; E-18) (Santa Cruz, sc-13454), goat polyclonal anti-SK1 (1:200; A-13) (Santa Cruz, sc-17991), goat polyclonal anti-eIF3η (1:200; A-20) (Santa Cruz, sc-16378), rabbit polyclonal anti-cleaved caspase-3 (1:100; Cell Signaling, #9664), mouse anti-PSD95 (1:200; Thermo Scientific), rabbit antisynapsin (1:1,000; Synaptic Systems), mouse anti-GFAP (1:200; Millipore), rabbit anti-Tbr1 (1:500, Abcam), rabbit anti-Dcx (1:100, Abcam).
NaAsO₂ was used at 200 μM (Sigma). The angiogenin small-molecule inhibitor N65828 (8-amino-5-(4′-hydroxybiphenyl-4-ylazo)naphthalene-2-sulphonate) was obtained from the National Cancer Institute (http://dtp.cancer.gov). NAC was purchased from Sigma and O-propargyl-puromycin (OP-puromycin) from Medchem Source LLP.

Cells on coverslips were washed twice with PBS (5 min each), fixed with 4% PFA at room temperature (RT) for 10 min, then rewashed twice with PBS (5 min each). Coverslips were incubated for 1 h at RT in PBS, 0.03% Triton X-100, 3% BSA and 2% normal donkey serum (NDS, Jackson laboratory, #017-000-121) for permeablization and blocking non-specific antibody binding. Following that, incubation of primary antibodies was often carried out overnight at 4°C in the same buffer. Cells were then washed (3 times, 10 min each) in the same buffer and incubated with secondary antibodies (Jackson Laboratory) for 1 h at RT. Cells were finally washed with PBS (3 times, 10 min each) and mounted on glass slide. Staining was viewed and analyzed with Olympus up-right fluorescent microscope (BX51) or with confocal microscope (Leica TCS SP5 II). The following antibodies were used: mouse anti-Oct4 (Millipore), goat anti-Sox2 (Santa Cruz), mouse anti-Nestin (Chemicon), mouse anti-MAP2 (Sigma), rabbit anti-Synapsin (Synaptic Systems), mouse anti-vGlut1 (Synaptic Systems), Rabbit anti-GABA (Sigma).