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- Origin of Ram Epididymal Fluid Exosome-Like Vesicles: MATERIALS AND METHODS(3)

After the centrifugation, a yellowish pellet was found at the bottom of the tube. This pellet was suspended in PBS and submitted to a second high-speed centrifugation. The final pellet gave a cloudy, refracting solution in PBS, which was divided into aliquots to be processed for electron microscopy or prepared for one- and two-dimensional gel separation.

Electron Microscopy

The washed pellet was fixed at room temperature with 2.5% glutaral-dehyde in 0.175 M cacodylate at pH 7.2. After 24 h, the pellet was postfixed with osmium tetroxide (2% final) and potassium ferricyanide (1.5%), washed with deionized water, and dehydrated with ethanol before embedding in epoxy-propan glycidether 100. The fixed pellet was cut with an ultramicrotome (80-90 nm). Contrast was enhanced by uranyl-acetate and lead citrate. Samples were observed on a Philips CM 10 microscope (60 KV). Measurement of the vesicle diameters and of the thickness of the vesicle membranes was performed either manually or using digital imaging system (Soft Imaging System, Munster, Germany). Both techniques gave very similar results.

February 14, 2014 Epididymal
Tags: epididymis epididymosomes exosomes gamete biology