Immunohistochemical Localization of nNOS in Dissociated Retinal Horizontal Cells From Goldfish Retina

Abstract

Abstract: : Purpose: In this study we investigated on the localization of nicotinamide adenine dinucleotide–diaphorase (NADPH–d) and neuronal nitric oxide synthase (nNOS) in isolated goldfish retinal horizontal cells (HCs). Methods: Isolated retinal cells were generally obtained by enzyme treatment from goldfish retina (Carassius auratus) adapted to normal light/dark adaptation. The cells were stained for NADPH–d activity by incubathing formaldehyde–fixed cells in PBS (pH 7.4) containing ß–NADH (1 mM) and 0.6 mM nitro blue tetrazolium for 3 hrs at 37 °C. For NOS immunohistochemistry formaldehyde–fixed cells were incubated overnight in PBS containing rabbit polyclonal anti–brain NOS antibody at 4 °C, and then incubated with Cy3 conjugated goat anti–rabbit IgG for 1 hr at room temperature. The Cy3–labeled cells were observed using fluorescence microscopy. Results: Positively activation by NADHP–d was shown in all types of cone–HCs and in rod–HCs. The H1 and H2 type cone–HCs also showed immunoreactivities to anti–nNOS in somata and their axon–terminals. The H3 type cone–HC and rod–HCs were not labeled by nNOS. Conclusions: These results indicate that H1 and H2 types of goldfish retinal horizontal cells could be the source of NO.

Keywords: horizontal cells • nitric oxide • immunohistochemistry