Ultramicrotome

For transmission electron microscopy (TEM), 1 × 10⁶ cells were seeded in culture dishes in a final volume of 5 mL. For PC12 cells, after centrifugation at 1000× g for 5 min, the cell pellet was rinsed in PBS and fixed for 90 min at 4 °C in a fixing solution containing 2.0% paraformaldehyde and 0.1% glutaraldehyde in 0.1 M PBS (pH 7.4). For SH-SY5Y cells, after removing culture medium, the fixing solution (2.0% paraformaldehyde/0.1% glutaraldehyde in 0.1 M PBS pH 7.4) was added to cell culture for 90 min at 4 °C. Cells were gently scraped from the plate and centrifuged at 10,000 rpm for 10 min to obtain the cell pellet.
After washing in PBS (0.1 M), both PC12 and SH-SY5Y specimens were post-fixed in 1% osmium tetroxide (OsO₄) for 1 h, at 4 °C, and they were dehydrated in increasing ethanol solutions (30%, 50%, 70%, 90%, and 95% for 5 min, and 100% for 60 min) to be embedded in epoxy resin.
Ultrathin sections (90 nm thick) were cut at ultra-microtome (Leica Microsystems, Wetzlar, Germany), counterstained with a saturated solution of uranyl acetate and lead citrate, dissolved in distilled water, and they were finally examined using a JEOL JEM SX100 transmission electron microscope (JEOL, Tokyo, Japan).

At the end of each study, two tissue wedges containing the TM and SC were dissected 180° apart from each anterior segment and fixed in 10% neutral buffered formalin. Tissue wedges were post fixed in 2% osmium tetroxide (Electron Microscopy Sciences, Hatfield, PA) in 0.1 M phosphate buffer followed by dehydration in ascending ethanol concentrations. Tissues were subjected to a clearing agent (acetone, Sigma-Aldrich), embedded in epoxy resin blocks, and 500 nm and 100 nm sections were obtained using an ultramicrotome (Leica Microsystems, Buffalo Grove, IL). 500 nm sections were stained with toluidine blue and gross morphology was assessed by light microscopy. 100 nm sections were placed on copper grids and stained with 2% uranyl acetate (Electron Microscopy Sciences) followed by lead citrate (Mager Scientific, Dexter, MI). Sections were imaged using a JEOL 1400 transmission electron microscope (JEOL USA, Peabody, MA) for evaluation of cell and tissue ultrastructure.

L. monocytogenes cells were fixed, sedimented and washed (0.05 M HEPES buffer, pH 7.2) as described above. Washed pellets were embedded in low melting point agarose (3%, mixed 1:1 with the sample; Sigma, Germany), trimmed into small pieces (about 1 × 1 mm) and fixed in 2.5% glutardialdehyde in 0.05 M HEPES buffer, pH 7.2. Samples were postfixed in osmium tetroxide (1% in water) for 60 min and 2 h in uranyl acetate (2% in water) at room temperature and dehydrated in an ethanol series (30, 50, 70, 90, 96% and absolute ethanol for 20 min). Samples were infiltrated with a mixture (1:1) of ethanol and resin (LR White hard grade; Science Services, Germany), embedded in pure resin and finally polymerized at 60°C overnight. Ultrathin sections of about 60–70 nm were cut with an ultramicrotome (Leica Microsystems, Germany), poststained with uranyl acetate followed by lead citrate. Sections were examined using a JEM-2100 transmission electron microscope (Jeol, Japan) at 200 KV. Images were recorded at full resolution of 2k × 2k with a Veleta side-mounted CCD camera (Olympus SIS, Germany).

Transmission electron microscopy was performed with young cells after a single freezing experiment at – 2 °C and – 10 °C and with old cultures (pre-akinetes) after freezing to – 20 °C and – 70 °C, plus the respective untreated controls. Chemical fixation of filaments was as described by Holzinger et al. (2009 (link)) with some modifications. Briefly, samples were fixed in 2.5% glutaraldehyde in 25 mM sodium cacodylate buffer (pH 6.8) for 1.5 h, post-fixed with 1% OsO₄ at 4°C for 12 h, rinsed and dehydrated in increasing ethanol concentrations, transferred via propylene oxide, and embedded in modified Spurr’s embedding resin (Science Services, Munich, Germany). Ultrathin sections were prepared with an Ultra-microtome (Leica Microsystems GmbH, Wetzlar, Germany), counterstained with 2% uranyl acetate and Reynold’s lead citrate, and investigated on a Zeiss LIBRA 120 transmission electron microscope (Carl Zeiss AG, Oberkochen, Germany) at 80 kV. Images were taken with a TRS 2 k SSCCD camera and processed with Adobe Photoshop 7.0 software (Adobe Systems Inc., San Jose, CA, USA).

After removal of the kidneys, the tissues were immediately diced into 1 mm³ pieces and fixed with 2.5% glutaraldehyde and 2% paraformaldehyde in sodium cacodylate buffer (pH 7.2) at 4 °C. Tissue specimens were then postfixed in 1% osmium tetraoxide (OsO₄) containing 1.5% potassium ferrocyanide for 30 min at 4 °C. The fixed tissues were dehydrated using an ethanol series (50%, 60%, 70%, 80%, 90%, and 100%) for 20 min at each concentration. The tissues were subsequently transferred to propylene oxide (Sigma-Aldrich) and embedding media (Spurr’s Kit; Electron Microscopy Science, Hatfield, PA). After impregnating pure resin, the tissue specimens were embedded in the same resin mixture and sectioned (60–70 nm) using an ultramicrotome (Leica Microsystems GmbH, Vienna, Austria). Then, they were double-stained with 2% uranyl acetate for 20 min and lead citrate for 10 min. The sections were then observed using a Hitachi H7600 transmission electron microscope (TEM) (Hitachi Ltd.) at 80 kV. The number of abnormal mitochondria was determined by randomly capturing 20 TEM micrographs per sample (image area = 8.5 µm², × 2000 magnification).

Approximately 10⁷ candidate HCC cells were harvested and fixed with 2.5% glutaraldehyde in phosphate buffer for 90 min. The cells were postfixed with 1% osmium tetroxide for 30 min followed by a gradient dehydration using ethanol and acetone and then embedded in Epon 812 resin. The 60-nm ultra-thin sections were obtained using an ultramicrotome (Leica Microsystems, Wetzlar, Germany) and placed on uncoated copper grids. The sections were stained with 3% lead citrate-uranyl acetate and then observed under the TECNAI20 electron microscope (Philips, Amsterdam, The Netherlands). The frequencies of autophagic vacuoles per cell were calculated by averaging the frequencies of autophagic vacuoles in 10 cells.