Calcium-Dependent Actin Filament: MATERIALS AND METHODS(1)

MATERIALS AND METHODS(1)

Animals

The bovine testes and epididymides used in this study were obtained immediately after death from the slaughterhouse Les Abattoirs Les Cedres (Sainte-Anne de Bellevue, PQ, Canada). We used 10 testes from normal 8- to 9-mo-old fetuses and 10 testes from 2-yr-old normal adults. The fetuses were obtained from gestating cows immediately after death. Adrenal glands and part of the ascending aorta and of the superior vena cava were also dissected. Cheap Diskus Advair

Isolation of Seminiferous Tubules and Other Tissue Preparations

Testes obtained immediately after death were decapsu-lated, and small pieces of tissue were placed in cold Minimum Essential Medium (MEM) (Gibco BRL, Oakville, ON, Canada). Testicular tissue pieces were incubated 2 X 45 min with 0.25 mg/ml collagenase D (Boehringer-Mann-heim, Laval, PQ, Canada) and 0.1 mg/ml soybean trypsin inhibitor (Sigma, St. Louis, MO) at 37°C in a water shaker bath set at 80 cycles/min. The reaction was stopped by addition of an equal volume of MEM. The seminiferous tubules were allowed to settle by gravity while interstitial cells occupied the supernatant. Further separation was achieved by centrifugation at low speed (400 rpm, GS-6R Beckman centrifuge; Beckman, Mississauga, ON, Canada) for 15 min. The two components of the testis bear distinct colors, thus making them easy to identify: the tubules are whitish and the interstitial tissue brownish. The tubules and the interstitial cells were quickly washed in PBS (137 mM NaCl, 3 mM KCl, 8 mM Na2PO4, 1.5 mM KH2PO4, pH 7.4) and homogenized with a glass tissue grinder in PBS containing 1 mM of PMSF (Sigma). Bovine chromaffin cells were isolated as previously described. Tissue fragments from the aorta and the vena cava were homogenized in PBS containing 1 mM PMSF with a Polytron tissue homogenizer (Brinkmann Co., Westbury, NY).

Category: Protein

Tags: calcium, Filament, Protein, Testis

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