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Rat Testis Motor Proteins Associated with Spermatid Translocation: MATERIALS AND METHODS(1)

MATERIALS AND METHODS(1)

Animals

Animals used in this study were mature Sprague-Dawley rats that ranged in weight from 240 g to 390 g. While animals were under deep halothane anesthesia, their testes were removed for fixation or epithelial isolation, and then the animals were killed. birth control yasmin

Reagents

Most reagents were obtained from Sigma-Aldrich (Oakville, ON, Canada) unless otherwise indicated. Fluorescent phallotoxins were obtained from Molecular Probes (Eugene, OR). Paraformaldehyde was purchased from Fisher Scientific (Vancouver, BC, Canada).

Immunological reagents were obtained from two sources. Monoclonal antibodies to the intermediate chain of cytoplasmic dynein (clone 74.1) and kinesin II (clone K2.4) were obtained from Berkeley Antibody Company (Berkeley, CA). The antibodies were stored at concentrations of 1.0 mg/ml and 5.0 mg/ml, respectively. Secondary antibodies for immunofluorescence were obtained from Jackson ImmunoResearch Laboratories Inc. (West Grove, PA) and consisted of goat anti-mouse IgG conjugated to either Texas Red or fluorescein.

Preparation of Tissue for Immunofluorescence

The spermatic arteries of isolated testes were cannulated using 26-gauge needles attached to a gravity-fed perfusion apparatus, and the organs were perfused for 1-2 min with warm (33°C) PBS (150 mM NaCl, 5 mM KCl, 3.2 mM Na2HPO4, 0.8 mM KH2PO4) and then perfused for 30 min with fixative (3% paraformaldehyde, PBS, pH 7.3, 33°C). After this, the testes were washed with PBS by perfusion for an additional 30 min.