Anti s100a9

Proteins (30 μg) from each sample were subjected to SDS-PAGE (Sodium Dodecyl Sulfate Polyacrylamide Gel Electrophoresis) (10% acrylamide, w/v) and transferred to PVDF (polyvinylidene fluoride) membranes (Amersham Biosciences, Uppsala, Sweden). Nonspecific binding sites were blocked with 5% (v/v) nonfat milk in 50 mM Tris-HCl, pH 7.4, containing 150 mM NaCl, 0.1% (w/v) Triton X-100 at 20°C at room temperature for 1 h. Immunological detection was performed using the anti-MMP9 antibody at 1:1000 dilution (Cell Signaling Technology, Inc, Danvers, MA, USA), anti-S100A9 at 1:250 dilution (HPA004193, Sigma-Aldrich), and anti-clusterin at 1:100 dilution (Santa Cruz Biotechnology, Santa Cruz, CA, USA). After washing three times, blots were further incubated with a horseradish peroxidase-conjugated secondary antibody (1: 5000 dilution) at room temperature for 1 h. Protein intensities were determined using densitometry (LabWorks, UVP, Inc., Upland, CA, USA). Differences with p values<0.05 were considered to be statistically significant.

An additional set of formalin-fixed and paraffin-embedded archival tissue specimens, composed of 50 cases of NNCM, 50 cases of ACP, 30 cases of CCIS, 63 cases of ICC, was obtained from the Department of Pathology of Xiangya Hospital at Central South University, used for immunohistochemical analysis according to the procedure described in our previous study [46 (link)]. Briefly, the sections were incubated with anti-TNC (1:1000; sigma) or anti-S100A9 (1:800; sigma) overnight at 4°C, and then were incubated with biotinylated secondary antibody followed by addition of avidin-biotin peroxidase. Diaminobenzidine was used as the chromogen. Finally, the sections were counterstained with hematoxylin. As a negative control, the primary antibody was omitted. The evaluation of immunostaining was performed as previously described by us [46 (link)]. A score (ranging from 0–6) was obtained for each case. A combined staining score of≤2 was considered to be negative staining (no expression); a score between 3 and 4 was considered to be moderate staining (low expression); and a score between 5 and 6 was considered to be strong staining (high expression).