Horseradish peroxidase conjugated anti rabbit antibody

For immunohistochemistry (IHC) analysis, the TMA sections were deparaffinized in 100% xylene and rehydrated in graded ethanol solutions. The sections were then boiled under pressure in citrate buffer (pH 6.0) for 5 minutes for antigen retrieval. TMA sections were incubated overnight with a primary anti-GRO-β antibody (Catalog 500-P104, PeproTech, Rocky Hill, NJ, USA) diluted 1 : 400 in TBS containing 1% bovine serum albumin. After washing, sections were incubated with anti-rabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA). GRO-β immunostaining was evaluated independently by two trained pathologists who were blinded to the clinical background of the cases. Positivity of cell staining was recorded as a percentage (0–100%).
The cutoff point for the GRO-β expression score that was statistically significant in terms of overall survival (OS) was determined using the X-tile software program (Rimm Lab, Yale University, New Haven, CT, USA), as described elsewhere [20 (link)]. The degree of staining was quantified using a two-level grading system, and staining scores were defined as follows: for GRO-β, 0–75 was regarded as low expression while 76–100 was regarded as high expression.

Western blot analysis was performed as described previously^{26 (link)}. The cells were washed in cold PBS twice and lysed in RIPA buffer (Thermo-Fisher Scientific, Roskilde, Denmark) supplemented with protease inhibitors (Roche, Hvidovre, Denmark) for 30 min in cold room. Samples were centrifuged at 12,000 r.p.m., 4 °C for 10 min. Protein concentrations were determined with a BCA kit (Thermo-Fisher Scientific, Roskilde, Denmark), and equal amounts (30 µg) of proteins were loaded on a polyacrylamide gel (Thermo-Fisher Scientific, Roskilde, Denmark). Blotted nitrocellulose membranes were incubated overnight with primary antibody at 4 °C, and were developed after 1 h incubation with secondary anti-rabbit horseradish peroxidase-conjugated antibody (Santa Cruz Biotechnology, Heidelberg, Germany) using an ECL Western blotting kit (GE Healthcare, Brøndby, Denmark) and Kodak films. Antibodies for KIAA1199 was purchased from ProteinTech, Manchester, UK; Antibodies (total or phosphor) specific for CFL1, LIMK1, p38, GSK3β and beta-catanin were obtained from Cell Signaling (Herlev, Denmark); antibodies for DSTN and actin were bought from Sigma-Aldrich (Copenhagen, Denmark). All antibodies were used at 1:1000 dilutions except actin antibodies (1:2500).