Ab105802

The expression of annexin A13 was examined by immunohistochemistry on FFPE sections using a rabbit polyclonal antibody against annexin A13 (ab105802, Abcam, USA). Annexin A13 expression was evaluated in both tumor and adjacent normal tissues. Tissue sections were dewaxed, rehydrated, and followed by heat-induced antigen retrieval. Endogenous peroxidase activity was removed by applying 3% H₂O₂ for 5 minutes. After blocking with 5% normal goat serum, primary antibody was applied overnight at 4°C, followed by HRP-tagged goat anti-rabbit IgG secondary antibody (sc-2030, Santa Cruz, USA). Immunosignals were then visualized using diaminobenzidine and slightly counterstained with hematoxylin. Sections were evaluated for intensity under light microscope (no staining = 0; light staining = 1; moderate = 2; strong = 3). Any IHC intensity greater than 0 was defined as IHC positive.

The following antibodies were used in the western blot experiments: ANXA13, rabbit polyclonal (ab105802, Abcam, USA; the specificity of this antibody was further verify by a second ANXA13 antibody: goat polyclonal, AF4149-SP, R&D System, USA); β-actin, mouse monoclonal (clone AC-74, A2228, Sigma); MMP9, rabbit polyclonal (ab38898, Abcam); Phospho-Akt (Thr308) (244F9), Rabbit mAb #4056 (Cell Signaling Technology). Protein lysates were prepared from cultured cells in ice-cold Tris buffer (20 mmol/L; pH 7.5) containing NaCl (137 mmol/L), EDTA (2 mmol/L), Triton X (1%), glycerol (10%), NaF (50 mmol/L), DTT (1 mmol/L), and a protease inhibitor cocktail (P8340, Sigma, USA). After electrophoresis (SDS-PAGE), protein samples were transferred onto Hybond-P membranes (Amersham Biosciences). The blot was blocked in TBS with 5% nonfat milk and 0.1% Tween 20, followed by incubation with primary antibody (RT 1h) and HRP-tagged goat anti-rabbit or -mouse IgG secondary antibody (sc-2030 or sc-2357, Santa Cruz, USA). The blots were visualized and quantitated using Amersham ECL Western Blotting Detection Kit (Amersham Biosciences).