Powergen 500

Aqueous polymer solutions were prepared freshly (20 mM) in sterile, normal saline and sonicated at room temperature until fully dissolved. Saline was composed of 0.9% (w/w) sodium chloride.
Paclitaxel/medium chain triglycerides (MCT) solutions were prepared freshly. Two milliliters of paclitaxel solution (prepared at a concentration of 7.5 mg/mL in 50:50, acetonitrile:ethanol) was dissolved in MCT followed by heating and stirring until fully solubilized. All traces of acetonitrile and ethanol were removed by vacuum.
Paclitaxel/MCT solution and PFCE were added to the polymer solution. The homogenizer and microfluidizer were first cleaned with 100% and 70% ethanol followed by 100% and 70% methanol and finally three rinses with Millipore Milli-Q water to remove all traces of any previous nanoemulsions. The prepared mixture was then homogenized with the high-speed homogenizer (Power Gen 500, Fisher Scientific, Hampton, NH) for 1 min at 21,000 rpm at room temperature. The resulting crude emulsion was then further mixed with the microfluidizer (model M-110S, Microfluidics Corp., Newton, MA) for 1 min under 5,000 psi with the cooling bath kept at 0 °C. The final emulsion was then filtered with a 0.45 μm nylon filter and stored in a sterile, plastic centrifuge tube (Corning Inc., Corning, NY) at 4 °C.

Intestinal tissue from 5-d-old pups was transferred into a pre-tarred
tube with 1 ml of sterile PBS. Tissue was homogenized using PowerGen500
(Fisher), serially diluted in sterile PBS and plated on BHI (Difco) and MRS
(Difco) agar overnight at 37 °C in a container with BD GasPakEZ
(anaerobic) or ambient air (aerobic). Dilutions with between 30 and 300 colonies
were recorded for colony counts after 24 h. Agar plates were assessed for
colonies with different colony morphologies. Over two dozen colonies from three
separate experiments were kept as isolates after additional re-streaking to
confirm single-colony identity. The whole 16S rRNA gene was then amplified from
each isolate using 500 nM 8F (5′-AGAGTTTGATCCTGGCTCAG-3′) and
1492R (5′-GGTTACCTTGTTACGACTT-3′) with Phusion high-fidelity
polymerase (NEB) under the following reaction conditions: initial denaturation
at 98 °C for 3 min; 34 cycles of denaturation at 98 °C for 30 s,
annealing at 53 °C for 30 s and extension at 72 °C for 30 s; and
final extension at 72 °C for 3 min. Amplicons from each reaction were
column purified (Qiagen) and subjected to Sanger sequencing spanning the entire
region. The consensus sequence for each isolate was then determined, and the
sequence was classified using BLAST against the NCBI 16S rRNA gene database
(with a sequence identity of 98–100%).

Following bioluminescence imaging, intestinal and extra-intestinal
organs were transferred to a pre-tarred tube with 1 ml of sterile PBS. Tissue
was homogenized using PowerGen500 (Fisher), serially diluted in sterile PBS, and
plated on MacConkey agar overnight at 37 °C in ambient air. Dilutions
with between 30 and 300 colonies were recorded for colony counts the following
morning. During homogenization, the homogenizer was cleaned with 70% ethanol and
water between samples, and both blank and uninfected organs were used as
negative controls.

Samples of ALD, AD, BC, FT, GH, HA, IO, LR, MH, MG, MR, and MT were prepared for electrophoretic identification of MHC. Approximately 40–50 mg of muscle tissue was cut from the frozen tissue block, homogenized in 200μl of 0.1M potassium phosphate (PBS) buffer (pH 7.3) and 5% protease inhibitor cocktail (Sigma, Aldrich) following Kohn and Myburgh^{23 (link)} with a tissue homogenizer (Fisher Scientific, PowerGen 500) in an ice bath, followed by centrifugation at 10,000g (4°C) for 10 minutes and re-suspended in 0.1M PBS buffer (pH 7.3) and 5% protease inhibitor cocktail for extraction of the myosin fraction. Total protein content was assayed by bicinchoninic acid assay according to manufacturer specifications (Synergy HT multimode microplate reader, Biotek Instruments, Inc., Pierce® BCA protein assay, Thermo Fisher Scientific Inc). Samples were stored at −80°C.