Fig. 4.

Neuropil of supraoptic nucleus of the hypothalamus double-labelled for nNOS (immunoprecipitate - long arrows) and ET-1 (immunogold-silver grains – short arrows). a A fragment of a double-labelled dendrite (likely to be a primary or varicose dendrite from a bipolar magnocellular neurone) shows abundance of neurosecretory granules (nsg), of which some are nNOS-positive (long arrows); short arrows point to immunoreactivity for ET-1. Note unlabelled axon profile (Ax) making synapse on the double-labelled dendrite. b A double-labelled axon terminal (At) making asymmetric synapse with unlabelled dendrite spine (ds). The axon terminal contains numerous small agranular synaptic vesicles, membrane of which is labelled with nNOS immunoprecipitate; co-localised immunogold-silver grains of ET-1 labelling (arrows) are also seen. Note that an adjacent axon profile (Ax) containing small agranular synaptic vesicles is unlabelled; m-mitochondria. Bars: 0.5 μm. Affinity-purified rabbit polyclonal nNOS antibody (SC-1025, Santa Cruz, USA), which does not cross react with NOS2 and NOS3 was used at 0.8μg/ml in the preembedding ExtrAvidin immunocytochemical method. The rabbit polyclonal ET-1 antibody to human/porcine ET-1 (Sigma, Poole, UK), which does not cross-react with big ET but may with ET-2 and ET-3 was used at 1:1,000 in the preembedding immunogold-silver labelling method as the second immunolabelling. Images A and B are modified from Mukherjee and Loesch, Histochem J 2002, 34:181-187 [Kluwer Academic Publishers], which is kindly acknowledged