Gel doc 1000 imager

HPMCs were harvested from culture dishes, and proteins were extracted using radioimmunoprecipitation assay buffer containing Halt protease inhibitor (Pierce). Immunoblotting was performed with primary antibodies against fibronectin, ST2 and β‐galactosidase (all from Santa Cruz Biotechnology); E‐cadherin, Snail, collagen 4, alpha‐smooth muscle actin (αSMA), p65, phosphorylated (p‐)p65, CCN1, GAPDH and IL‐33 (all from Abcam); and β‐actin (Sigma‐Aldrich). Equal amounts (30 μg) of extracted protein were separated by 10% sodium dodecyl sulphate‐polyacrylamide gel electrophoresis and transferred to an Immobilon‐FL 0.4‐μm polyvinylidene difluoride membrane (Millipore). Horseradish peroxidase‐conjugated anti‐rabbit and antimouse IgG (both from Cell Signaling Technology) were used as secondary antibodies. Labelled proteins were detected by enhanced chemiluminescence (Amersham Pharmacia Biotech; ECLTM PRN 2106) using a Gel Doc 1000 imager with Multi‐Analyst v.1.1 software (Bio‐Rad).

PFGE analysis was performed on F. tularensis samples [18] (link). Agarose plugs were sliced and incubated in 10 U of restriction enzyme PmeI (Biolabs, New England) for 3 hours at 37°C. Electrophoresis was performed in 1% agarose with a switch time of 1.79 to 10.71 s at 6 V/cm for 24 hours at 14°C. Salmonella enterica serotype Braenderup strain H9812 restricted with XbaI was used for gel normalization. Gels were stained with gel red and gel images captured by using a Gel Doc 1000 imager (Bio-Rad).
PFGE images were analysed using Bionumerics v 6.01 (Applied Maths, Sint-Martens-Latem, Belgium). Unique PFGE patterns were analysed and compared manually for band polymorphism.