Em stainer

According to our previous report [17 (link)], samples were fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, then washed in 0.1 M sodium cacodylate buffer and treated with 0.1% Millipore-filtered cacodylate buffered tannic acid, postfixed with 1% buffered osmium, and stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium. The samples were polymerized in a 60 ℃ oven for approximately 3 days. Ultrathin sections were cut in a Leica Ultracut microtome (Leica, Deerfield, IL), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a JEM 1010 transmission electron microscope (JEOL, USA, Inc., Peabody, MA) at an accelerating voltage of 80 kV. Micrographs were taken at 7500 × or 50,000 × magnification.

A. salmonicida stains harboring plasmids pHSG299 or pHSG299‐mcr‐3 were cultured to log‐phase in tryptic soy broth (TSB) medium. Bacterial cells were then collected by centrifugation at 2000 × g and washed once with PBS. Cells were fixed in 2.5% (v/v) glutaraldehyde solution at 4 °C for 24 h. In preparation for TEM analysis, samples were embedded in Spur epoxy resin medium and polymerized at 70 °C for 3 days. Ultrathin sections were subsequently cut using a Leica Ultracut microtome (UC6i; Leica, Wetzlar, Germany) and stained with uranyl acetate and lead citrate in a Leica EM Stainer. The sections were examined using a JEOL JEM‐1230 transmission electron microscope (JEOL Ltd., Tokyo, Japan).

For transmission electron microscopy (TEM), samples were fixed with a solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer, pH 7.3, washed in 0.1 M sodium cacodylate buffer, treated with 0.1% Millipore-filtered cacodylate-buffered tannic acid, post-fixed with 1% buffered osmium, and then stained en bloc with 1% Millipore-filtered uranyl acetate. The samples were dehydrated in increasing concentrations of ethanol, filtered, and embedded in LX-112 medium. The samples were polymerized in a 60°C oven for approximately 3 days. Ultrathin sections were cut in an Ultracut microtome (Leica, Deerfield, IL), stained with uranyl acetate and lead citrate in an EM Stainer (Leica), and examined using a JEM 1010 transmission electron microscope (JEOL USA, Inc.) at an accelerating voltage of 80 kV. Digital images were obtained using an AMT Imaging System (Advanced Microscopy Techniques Corp., Danvers, MA).

K7M3, LM7, and CCH-OS-D cells were treated with 1μM GCB for 48 hours. Next, samples were fixed with a solution of 3% glutaraldehyde plus 2% paraformaldehyde in 0.1M cacodylate buffer, with a pH of 7.3. After fixation, the samples were washed in 0.1M cacodylate buffer and treated with 0.1% Millipore-filtered buffered tannic acid. The samples were then postfixed with 1% buffered osmium tetroxide for 30 minutes and stained with 1% Millipore-filtered uranyl acetate. Samples were washed several times in water, then dehydrated in increasing concentrations of ethanol, after which they were infiltrated and embedded in LX-112 medium. The samples were polymerized in an oven at 60°C for 2 days. Ultrathin sections were cut using a Leica Ultracut microtome (Leica), and these sections were stained with uranyl acetate and lead citrate using a Leica EM stainer and examined using a JEM 1010 transmission electron microscope (JEOL, USA, Inc.) at an accelerating voltage of 80 kV. Digital images were obtained using the AMT Imaging System (Advanced Microscopy Techniques Corp).

Cells were seeded at (1.0–2.5 × 10⁵ cells/well) in a 6-well plate and incubated for different intervals in amino acid-free medium. Samples were then fixed with Karnovsky’s fixative solution containing 3% glutaraldehyde plus 2% paraformaldehyde in 0.1 M cacodylate buffer at pH 7.3 and stored at 4 °C. After fixation, samples were submitted to the MD Anderson electron microscopy core facility for processing. Briefly, cells were washed in 0.1 M cacodylate buffer and treated with 0.1% Millipore-filtered buffered tannic acid, postfixed with 1% buffered osmium tetroxide for 30 min, and stained with 1% Millipore-filtered (0.2 μM) uranyl acetate. The samples were washed several times in water and then dehydrated in increasing concentrations of ethanol, infiltrated, and embedded in LX-112 medium. The samples were polymerized in a 60 °C oven for 2 days. Ultrathin sections were obtained using a Leica Ultracut microtome (Leica, Wetzlar, Germany), stained with uranyl acetate and lead citrate in a Leica EM Stainer, and examined in a JEM 1010 transmission electron microscope (JEOL USA Inc., Peabody, MA, USA) at an accelerating voltage of 80 kV. Digital images were obtained using AMT Imaging System (Advanced Microscopy Techniques Corp, Woburn, MA, USA).