Gatan ultrascan 1000 digital camera

Embryos were fixed in 0.1 M sodium cacodylate buffer (pH 7.4) with 2% glutaraldehyde and 1% paraformaldehyde, and post-fixed in the same buffer with 2% osmium tetroxide, then stained with 2% aqueous uranyl acetate, dehydrated in acetone, infiltrated and embedded in LX-112 resin (Ladd Research Industries). Samples were ultrathin sectioned on a Reichert Ultracut S ultramicrotome and counter stained with 0.8% lead citrate. Grids were examined on a JEOL JEM-1230 transmission electron microscope (JEOL USA) and photographed with the Gatan Ultrascan 1000 digital camera (Gatan).

To confirm fibrillation of synthetic amyloids, samples (500 µg/ml) were prepared with parlodion-filmed carbon-coated grids and 2% potassium phosphotungstate, pH6.5, and analyzed and photographed as previously described (Roan et al., 2009 (link)). To visualize the physical interaction between sperm cells and the fibrils, swim-up spermatozoa isolated as described above were incubated in the absence or presence of 100 µg/ml SEM fibrils for 1 hr, and then fixed in a 0.1 M sodium cacodylate buffer solution (pH 7.4) containing 2% glutaraldehyde. The samples were then loaded into 200 µm diameter cellulose capillary tubes (Leica Microsystems Inc., Buffalo Grove, IL), post-fixed in 2% osmium tetroxide in the same buffer, stained en block with 2% aqueous uranyl acetate, dehydrated in acetone, infiltrated, and embedded in LX-112 resin (Ladd Research Industries, Burlington, VT). Samples were ultrathin-sectioned on a Reichert Ultracut S ultramicrotome and counter-stained with 0.8% lead citrate. Grids were examined on a JEOL JEM-1230 transmission electron microscope (JEOL USA, Inc., Peabody, MA) and photographed with the Gatan Ultrascan 1000 digital camera (Gatan Inc., Warrendale, PA).