Anti s100a4 antibody

Cell lysate or human lung proteins (30 μg) were separated by 12.5% sodium dodecyl sulfate-polyacrylamide gel electrophoresis and transferred to polyvinylidene difluoride membranes (Millipore Corporation, Billerica, MA). The membranes were blocked in 5% skim milk (BD, Sparks Glencoe, MD) in Tris-buffered saline (TBS) containing 0.1% Tween 20 (Sigma-Aldrich, St. Louis, MO) for 1 h at room temperature. The membranes were incubated with a polyclonal anti-S100A4 antibody (1:1000, Abcam Cambridge, MA) overnight at 4 � C. Next, the membranes were incubated with horseradish peroxidase-conjugated secondary antibodies (Thermo Fisher Scientific, Inc., Rockford, IL). Detection was performed using Western Blotting Luminol Reagent (Santa Cruz Biotechnology, CA). Relative protein levels were determined by quantitative densitometry using Image J software (NIH, Bethesda, MD) with normalization to that of β-actin (Sigma-Aldrich).

Lung tissue sections from IPF patients were fixed in 4% buffered paraformaldehyde and embedded in paraffin. The tissue was cut into 4mm-thick slices, and the sections were deparaffinized and rehydrated in increasing concentrations of ethanol. The sections were treated with 1.4% H 2 O 2 -methanol for 30 min to block endogenous peroxidase, and non-specific binding was next blocked by incubation in 1.5% normal house serum. The sections were incubated with a monoclonal anti-alpha smooth muscle actin antibody (1:50, Abcam, Cambridge, MA) and polyclonal anti-S100A4 antibody (1:50, Abcam), followed by the ABC Kit (Vector Laboratories, Burlingame, CA). Color was developed using a Liquid DAB Substrate Kit (Golden Bridge International, Inc., Mukilteo, WA) and counterstained with Harris' hematoxylin (Muto Pure Chemicals Co., Ltd., Tokyo, Japan).