• 17
    Nov
  • Acrosome Biogenesis in the Hamster: MATERIALS AND METHODS(4)

Immunoblot Staining

Protein patterns on Western blots were visualized using Coomassie blue dye or colloidal gold . Immu-noblots were incubated in 5% goat serum, 2.5% BSA, 0.1% Tween 20 in PBS (150 mM NaCl, 20 mM sodium phosphate, pH 7.6) to block nonspecific binding sites. After three rinses in PBS containing 0.05% Tween 20 and 1% goat serum (PBS-Tw-GS), blots were incubated in primary antibody diluted in PBS-Tw-GS; controls received equivalent dilutions of nonimmune serum or normal ascites fluid. After three washes in PBS-Tw-GS, the blots were incubated in an affinity-purified horseradish peroxidase-conjugated secondary antibody (KPL Inc., Gaithersburg, MD) diluted in PBS-Tw-GS. Immunoreactive bands were visualized using diaminobenzidine for color development. buy asthma inhaler

Immunofluorescence

Cells were fixed for 30 min on ice with 4% formaldehyde in 0.1 M sodium phosphate, pH 7.4, and then per-meabilized for 30 min by dilution into an equal volume of fixative containing 0.2% Triton X-100. In some experiments, spermatozoa were permeabilized either by incubation in 0.1% Triton X-100 in TNI for 30 min at 4°C or by nitrogen cavitation at 400 psi for 10 min before fixation. After adhering to poly-L-lysine-coated coverslips, cells were rinsed in PBS and blocked with PBS containing 5% goat serum and 2.5% BSA.

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