Anti huc d

Whole-mount and section in situ hybridization were performed as previously described (Hou et al., 2013 (link)). Chick Cotl1 subclones (Hou et al., 2009 (link)) were linearized and digoxigenin (DIG)-labeled antisense RNA was generated (Stratagene).
The following primary antibodies were used for immunohistochemistry: anti-Coactosin (Sawady Technology, Tokyo, Japan), which was raised in rabbits using the bacterially expressed peptide NH2-DHKELDEDYIKNELK-COOH as previously described (Hou et al., 2009 (link)); anti-HuC/D (Molecular Probe); anti-neurofilament (3A10; Developmental Studies Hybridoma Bank [DSHB]), anti-Islet1/2 (2D6; DSHB); anti-cofilin (Sigma); anti-p-cofilin (Ser3) (sc-21867-R; Santa Cruz Biotechnology); anti-HA (3F10; Roche Applied Science); and anti-GFP (Invitrogen). Alexa-conjugated anti-mouse, anti-rat, and anti-rabbit antibodies were used as secondary antibodies (Invitrogen).
After SIM imaging, immunostaining was carried out as previously described (Nozumi et al., 2009 (link)). Cells were fixed in 1% glutaraldehyde in PBS (137 mM NaCl, 2.7 mM Na₂HPO₄, 8.1 mM KCl, and 1.5 mM KH₂PO₄), permeabilized with 0.1% Triton X-100 in PBS, and reacted with primary and secondary antibodies. Rhodamine-phalloidin was diluted in the secondary antibody solution. For glutaraldehyde fixation, background staining was reduced by treatment with 1% sodium tetrahydroborate.

Neuro-2a (mouse neuroblastoma) cells were lysed and total protein content was determined by measuring its absorbance. Aliquots of 50 μg total protein from each sample were subjected to SDS-polyacrylamide gel electrophoresis on a NuPAGE 4-12% Bis-Tris gel (Invitrogen, Carlsbad, CA) followed by transfer to a nitrocellulose membrane. The membrane was probed with primary antibodies anti-HuC/D (6ug/mL, Molecular Probes, Eugene, OR) followed by incubation with appropriate HRP-conjugated secondary antibodies (GE healthcare, Piscataway, NJ). The signal was visualized through the use of the ECL Plus Western Blotting Detection System (GE Healthcare) and exposure to ECL Hyperfilm (GE Healthcare). The membrane was stripped with Re-Blot Plus Strong (Chemicon, Temecula, CA).