Localization of DOR and evaluation of mitochondrial activity in equine sperm cells by fluorescence and confocal laser scanning microscopy. (A) Reactivity staining (green fluorescence) detected by the rabbit anti-DOR antibody is observed exclusively on the mid piece of the sperm tail; sperm cells were counterstained by Evans blue to visualize sperm morphology. Picture was captured by Nikon coolpix 990 digital camera. (B) The highly selective fluorescent tripeptide analogue of the DOR antagonist DMT-TIC detected DOR on the same sperm region; image captured by confocal laser scanning microscope. (C), (D) negative controls for the antibody and the fluorescent tripeptide: in (C), the primary antibody was omitted, only the secondary antibody-FITC conjugated was employed; in (D) spermatozoa were incubated in the presence of 0.2 μmol l-1 naltrindole to block binding sites for the tripeptide. (E) The mid piece of an equine sperm cell containing active mitochondria was intensively labelled by MitoTracker orange (arrow), the cell with inactive mitochondria was only faint coloured in the same region (double arrow). Scale bars, 9.0 μm.