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.
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.