8-megapixel digital camera

For negative staining and analysis by transmission electron microscopy, bacterial suspensions in PBS were allowed to absorb onto freshly glow-discharged Formvar/carbon-coated copper grids for 10 min. Grids were washed in distilled H₂O and stained with 1% aqueous uranyl acetate (Ted Pella, Inc., Redding, CA) for 1 min. Excess liquid was gently wicked off, and grids were allowed to air dry. Samples were viewed on a JEOL 1200EX transmission electron microscope (JEOL United States, Peabody, MA) equipped with an 8-megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA).

For negative staining and analysis by TEM, bacterial suspensions in PBS or OMV were allowed to absorb onto freshly glow-discharged Formvar/carbon-coated copper grids for 10 min. The grids were washed in distilled water and stained with 1% aqueous uranyl acetate (Ted Pella, Inc., Redding, CA) for 1 min. Excess liquid was gently wicked off, and the grids were allowed to air dry. The samples were viewed on a 1200EX transmission electron microscope (JEOL USA, Peabody, MA) equipped with an 8-megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA).

For negative staining and analysis by TEM, bacterial suspensions in PBS or OMV were allowed to absorb onto freshly glow-discharged Formvar/carbon-coated copper grids for 10 min. The grids were washed in distilled water and stained with 1% aqueous uranyl acetate (Ted Pella, Inc., Redding, CA) for 1 min. Excess liquid was gently wicked off, and the grids were allowed to air dry. The samples were viewed on a 1200EX transmission electron microscope (JEOL USA, Peabody, MA) equipped with an 8-megapixel digital camera (Advanced Microscopy Techniques, Woburn, MA).

Samples were fixed in 2% paraformaldehyde/2.5% glutaraldehyde (Polysciences) in 100 mM sodium cacodylate buffer, pH 7.2, for 1 hour at room temperature. Samples were washed in sodium cacodylate buffer and postfixed in 1% osmium tetroxide (Polysciences) for 1 hour. Next, samples were rinsed extensively in dH₂O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella) for 1 hour. Following several rinses in dH₂O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA) equipped with an AMT 8 megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques).

Tumor tissues from Con and FF livers that had been intracardially injected with Bo1 cells for 10 days were cut into about 2 mm size and fixed at 5% glutaraldehyde in 0.16 M collidine buffer overnight. Then samples were washed in sodium cacodylate buffer at room temperature and postfixed in 1% osmium tetroxide (Polysciences Inc.) for 1 hour. Samples were then rinsed extensively in distilled H₂O (dH₂O) before en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) for 1 hour. Following several rinses in dH₂O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc.), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc.) equipped with an AMT 8-megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques).

For ultrastructural analyses, infected HFF cells were fixed in a freshly prepared mixture of 1% glutaraldehyde (Polysciences Inc.) and 1% osmium tetroxide (Polysciences Inc.) in 50 mM phosphate buffer at 4°C for 30 min. The low osmolarity fixative was used to dilute soluble cytosolic proteins and enhance the visualization of mitochondria. Samples were then rinsed extensively in cold dH₂O prior to en bloc staining with 1% aqueous uranyl acetate (Ted Pella Inc.) at 4°C for 3 hr. Following several rinses in dH₂O, samples were dehydrated in a graded series of ethanol and embedded in Eponate 12 resin (Ted Pella Inc.). Sections of 95 nm were cut with a Leica Ultracut UCT ultramicrotome (Leica Microsystems Inc.), stained with uranyl acetate and lead citrate, and viewed on a JEOL 1200 EX transmission electron microscope (JEOL USA Inc.) equipped with an AMT 8-megapixel digital camera and AMT Image Capture Engine V602 software (Advanced Microscopy Techniques). For cristae enumeration, images were blinded and the mitochondrial area along with the corresponding number of cristae in each area were counted using FIJI. The images were subsequently un-blinded and the values for rapamycin-treated and vehicle were compared using a Mann-Whitney test.