Image capture engine v602

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.