Uranyl acetate

The extensor digitorum longus muscle was excised and fixed under tension in modified Karnovsky fixative containing 2.5% glutaraldehyde (Electron Microscopy and Sciences, Hatfield, PA) and 2.5% formaldehyde (EMS) in sodium cacodylate (Sigma-Aldrich) buffer, pH 7.4. Tissues were fixed for 1h at room temperature and then cut in fixative lengthwise into 300 μm length x 300 μm height tissue pieces. Tissues were washed with cacodylate buffer and then transferred to a 300 μm deep aluminum planchette (Technotrade Int., Manchester, NH). The planchette was filled with 20% BSA (Sigma-Aldrich) in Gomori phosphate buffer, pH 7.4 containing 5% FBS (GIBCO/ThermoFisher), capped, and the tissue frozen in a Wolwhend Compact 02 High Pressure Freezer (Technotrade Int.). The frozen tissues were embedded in LX112 resin (Ladd Research, Williston, VT) after super-quick freeze substitution (Reference, below) in 1% osmium tetroxide (Ted Pella Inc., Redding, CA), 0.1% uranyl acetate (Ted Pella), and 5% dH₂O in acetone. Sections were cut using an Ultracut E Ultramicrotome (Leica Microsystems, Buffalo Grove, IL), placed on formvar and carbon-coated slot grids, and examined in a JEOL 1400 electron microscope (JEOL, Peabody, MA) outfitted with Orius CCD cameras (Gatan, Pleasanton, CA). Digital Micrograph software (Gatan) was used to collect digital images.

E. coli MG1655 and MG1655-pFadR were cultured at 37°C in LB-0.2% glucose from a single colony to OD₆₀₀ = 0.15–0.3 with shaking (200 rpm). Cultures were then back-diluted into fresh medium to an OD₆₀₀ of 0.005, at which point MG1655-pFadR samples were induced with the indicated concentrations of IPTG, cultured to OD₆₀₀ = 0.15–0.3, and then fixed. 1 ml of culture was pelleted for 5 min at 5,000 × g, washed once in 1X PBS, then resuspended in fixative (3% PFA/1% tannic acid/100mM cacodylate buffer) and incubated for 1 h with gentle rocking at room temperature. After fixation, samples were washed 3 times over the course of a few hours. Secondary fixation was performed with 1% osmium tetroxide/16.5 mg/ml potassium ferricyanide/100 mM cacodylate buffer for 1 h at room temperature, then washed 3 times in dH2O and stored overnight at 4°C. Samples were then en bloc stained with 1% aqueous uranyl acetate (Ted Pella Inc., Redding, CA) for 1 hr. Following several rinses in dH₂0, 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., Bannockburn, IL), then stained with uranyl acetate and lead citrate (Ted Pella Inc.).