Fig. 2. Immunochemical investigation of cGMP formation and the expression of NOS in the embryonic enteric nervous system. (A,B) Confocal fluorescence images of migrating enteric neurons on the midgut at 65% E in a so-called `tissue blot' preparation. Neurons were marked immunocytochemically for anti-cGMP (red) and anti-acetylated -tubulin (green). (C) Merged image of A and B. m, gut musculature. Scale bar: 50 µm. (D) Immunoblot analysis with an antibody against universal NOS in homogenates of different parts of embryonic gut and in the CNS (65-70% E). The antibody recognizes a protein of 135-140 kDa and a second, somewhat lighter protein band in the fore- and midgut cytosolic fraction. Bottom lanes provide an acetylated -tubulin band as loading control. For quantification, the ratio of the NOS signal to the -tubulin signal was calculated and averaged for all probed blots (mean NOS ratios, n=4, s.e.m. of the ratios are: ±0.07 MG, ±0.04 MG-M, ±0.01 FG, ±0.08 CNS). The NOS ratio directly below the lanes corresponds to the actual signals of the shown western blot. MG, midgut; MG-M, membranous part of midgut (both midgut homogenates included adjacent hindgut); FG, foregut; CNS, central nervous system, including ventral nerve cord.