Cellulose capillary tubes

Samples for electron tomography were prepared as described (Woog et al., 2012 (link)). Briefly, hermaphrodites were dissected in Minimal Edgar’s Growth Medium (Edgar, 1995 (link)) and embryos in early meiosis were selected and transferred to cellulose capillary tubes (Leica Microsystems, Vienna, Austria) with an inner diameter of 200 μm. The embryos were observed with a stereomicroscope, transferred to membrane carriers at appropriate stages, and immediately cryo-immobilized using an EMPACT2 high-pressure freezer (Leica Microsystems) equipped with a rapid transfer system (Pelletier et al., 2006 (link)). Freeze substitution was performed over 3 days at −90°C in anhydrous acetone containing 1% OsO₄ and 0.1% uranyl acetate using an automatic freeze substitution machine (EM AFS, Leica Microsystems). Epon/Araldite-infiltrated samples were then embedded in a thin layer of resin and polymerized for 3 days at 60°C. Embedded embryos were re-mounted on dummy blocks and serial semi-thick (300 nm) sections were cut using an Ultracut UCT Microtome (Leica Microsystems). Sections were collected on Formvar-coated copper slot grids and post-stained with 2% uranyl acetate in 70% methanol followed by Reynold’s lead citrate.

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).