Fig. 1. Immunocytochemistry, trituration loading, and specificity of L1 and NCAM antibodies in chick DRG neurons.A, Specificity of 8D9, 4d, and 5e is demonstrated by Western blot analysis. Lane 1, DRG lysate probed with 8D9. Two bands are observed, likely because of proteolysis. Lane 2, DRG lysate probed with 4d recognizes only NCAM-180;lane 3, purified NCAM-180 probed with 4d; lane 4, whole chick brain lysate probed with 4d; lane 5, DRG lysate probed with 5e, which recognizes all three NCAM isoforms. Note that different gels were used for lane 1 and for lanes 2–5 so that the migration distances do not correspond between these lanes. B,Trituration loading of intra-L1 followed by fixation and staining with secondary antibody (L1, Loaded) results in a similar pattern to that observed by immunocytochemistry with intra-L1 (L1, Intra) and with 8D9, which shows expression of L1 in growth cones (L1, Extra). Trituration loading of 4d followed by fixation and staining with secondary antibody (NCAM, Loaded) shows similar specific staining compared with indirect immunocytochemistry using 4d (NCAM, Intra) or with 5e (NCAM, Extra).