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. 2008 Jan 10;586(Pt 7):1823–1832. doi: 10.1113/jphysiol.2007.149450

Figure 4. Kv7.4 channels are expressed in serotonergic (5-HT) neurons in the rat DRN.

Figure 4

Upper panels (40× magnification, 40 μm section): Confocal scanning of Kv7.4 and 5-HT immunoreactivity shows an overlap of Kv7.4 channel and 5-HT expression in a subpopulation of neurons in the rat dorsal raphe nucleus (DRN). Lower panels (40× magnification, 40 μm section): Representative retrograde tracing experiment showing that Kv7.4 channels colocalize with rhodamine-coupled latex microsphere retrobeads (Lumaflour, Naples, FL, USA) pressure-injected unilaterally into the rat hippocampus, indicating that Kv7.4 channels are expressed in several DRN neurons projecting to the hippocampus. After 10 days the rat was perfused and 40 μm DRN sections processed for double immunohistochemistry. A rabbit polyclonal Kv7.4 channel (Kharkovets et al. 2000) diluted 1: 5000 and mouse monoclonal 5-HT antibody (1: 5000; DiaSorin, Saluggia, Italy) were used. The secondary antibodies were either Cy3-conjugated sheep anti-mouse IgG (1: 200) or Alexa 633-conjugated donkey anti-mouse/goat IgG (1: 200) mixed with biotinylated donkey anti-rabbit IgG (1: 800) (all from Jackson ImmunoResearch Laboratories, West grove, PA, USA). The immunoreactions were completed using the biotinylated tyramine procedure as previously described together with Fluorescein Avidin D (1: 200; Vector Laboratories, Burlingame, CA, USA) as previously described (Hansen et al. 2006).