01 Feb

Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: MATERIALS AND METHODS(8)

Confocal Laser Scanning Microscopy

Ethanol-permeabilized and air-dried sperm smears were stained sequentially with the three fluorescent markers, FITC-Pisum sativum, TOTO-3 iodide, and Nile red (Molecular Probes, Eugene, OR) because their targets are different (acrosome, nucleus, and plasma membrane of per-meabilized sperm, respectively). Samples were examined under a Bio-Rad MRC-1024 Laser Scanning Confocal Microscope (Bio-Rad Laboratories, Hercules, CA) equipped with a krypton/argon mixed gas laser (excitation lines 488, 568, and 647 nm) and mounted on a Nikon Eclipse E800 (Garden City, NY) series upright microscope. levitra super active plus

The fluorescence emissions of fluorescein, TOTO-3 iodide, and Nile red from the acrosomal region, nucleus, and the plasma membrane of sperm after ethanol permeabilization were simultaneously detected using the 598/40 nm, 522 DF32, and 680 DF32 emission/filter, respectively. Confocal images were obtained using a Nikon X60 (NA 1.35) objective lens and Kalman collection filter. Digitized images were saved on a Jaz disk (Iomega Corporation, Roy, UT) and processed with the Adobe Photoshop software (Adobe Systems, Mountain View, CA). Final images were printed using a Fujix Pictography 3000 (Fuji Photo Film Co., Tokyo, Japan) color printer.

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