Mouse igg2b anti huc d

Immunostaining was performed as described (Nikitina et al., 2009 ) with slight modifications; embryos were incubated overnight at 4 °C in primary antibody in blocking solution (10% sheep serum in PBS with 0.1% Triton X-100); secondary antibodies were also incubated overnight at 4 °C. Histochemical reactions were performed as described (Patel, 1994 (link)). Before imaging, embryos were cleared through a glycerol series into 70% glycerol in PBS. Primary antibodies: 1:50 HNK1 (mouse IgM, clone 3H5, Developmental Studies Hybridoma Bank); 1:500 anti-HuC/D (mouse IgG2b; Invitrogen); 1:200 anti-neurofilament-M (mouse IgG2a; Invitrogen); 1:200 anti-Pax3/7 (clone DP312; Davis et al., 2005 (link)). (The Developmental Studies Hybridoma Bank was developed under the auspices of the NICHD and is maintained by the University of Iowa, Department of Biological Sciences, Iowa City.) Secondary antibodies: 1:1000 Alexa⁴⁸⁸-conjugated goat anti-mouse IgG and/or Alexa⁵⁹⁴-conjugated goat anti-mouse IgG (Invitrogen), or 1:600 horseradish peroxidase-conjugated or alkaline phosphatase-conjugated goat anti-mouse IgG (Jackson ImmunoResearch).