02 Feb

Aryl Phosphate Derivatives of Bromo-Methoxy-Azidothymidine: MATERIALS AND METHODS(9)

MATERIALS AND METHODS(9)

High-Resolution Low-Voltage Scanning Electron Microscopy (HR-LVSEM)

HR-LVSEM was utilized for topographical imaging of different membrane domains over the sperm head as described previously. Aliquots (20 X 106) of motile sperm were incubated with DMSO alone (1%), or 100 ^M each of WHI-05 or WHI-07 or 10 |xM CaI in DMSO for 3 h at 37°C. Washed sperm suspensions were placed on 0.1% poly-L-lysine-coated glass chips and allowed to adhere to the glass over a 60-min incubation period on ice. The supernatants were decanted, and adherent cells were fixed in 1% paraformaldehyde and 1% glutaraldehyde in 0.14 M sodium cacodylate buffer for 3 h. Buy Asthma Inhalers Online

To preserve plasma membrane integrity, sperm were postfixed in 1% osmium tetroxide (OsO4) containing 0.1% ruthenium red in 0.14 M cacodylate buffer for 1 h at 4°C. All samples were dehydrated through an ascending ethanol series, critical point dried, and coated with approximately 2 nm of platinum using ion beam sputtering with argon (4 mA at 10 kV; Ion Tech Ltd., Middlesex, England). All samples were examined in a Hitachi S-900 SEM (Hitachi Instruments, Gaithersburg, MD) at an accelerating voltage of 2 kV. Sperm were observed under low magnification (X2000-5000), and representative sperm were photographed under intermediate magnification (X18 000-25 000). In each specimen evaluated, at least 200 sperm were scanned for the intactness of sperm acrosomal region.

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