Biotinylated goat anti mouse antibody

The protein expression of adhesion molecules was examined by EIA as described previously [5 (link)]. Briefly, HUVEC monolayers in 96-well plates were fixed by the addition of 4% paraformaldehyde. To create a standard curve, each 96-well plate was coated with diluted capture antibody (R&D Systems, Minneapolis, MN, USA), and standards were added according to the manufacturer’s instructions. Mouse anti-E-selectin monoclonal, mouse anti-P-selectin monoclonal, mouse anti-ICAM-1 monoclonal, and mouse anti-VCAM-1 monoclonal antibodies (1 μg/mL; R&D Systems) were added to each plate. The wells were washed twice with wash buffer, and biotinylated goat anti-mouse antibody (Dako, Carpinteria, CA, USA) was added. The wells were again washed twice with wash buffer, followed by the addition of streptavidin-horseradish peroxidase complex (Dako). After two washes with wash buffer, O-phenylenediamine dihydrochloride in citric acid phosphate buffer (pH 5.0) was added and reacted for 5 minutes at room temperature. The reaction was stopped by the addition of 1 mol/L sulfuric acid. Absorbance was measured at 490 nm using an enzyme-linked immunosorbent assay reader (Model 550; Bio-Rad, Hercules, CA, USA).

Samples from ectopic lesions were formalin fixed for 4 h and embedded in paraffin. Samples were sectioned at 10 μm, mounted on glass slides, dewaxed in xylene, and rehydrated through a series of increasing ethanol concentrations. Non-specific binding was blocked by incubation in 3% bovine serum albumin (BSA) in tris-buffered saline (Tris 100 mM, NaCl 0.15 M; pH7.4) for 30 min. Sections were incubated with either mouse anti-cytokeratin antibody (Novus Biologicals, Littleton, CO, USA, 1:200 dilution) or a rabbit anti-CD10 antibody (Novus Biological, 1:100 dilution) diluted in tris buffer containing 3% BSA in a humidified chamber overnight. Slide were washed with tris-buffered saline containing 0.1% Tween 20 and incubated with either biotinylated goat anti-mouse antibody (Dako, Glostrup, Denmark, dilution 1:200) or swine anti-rabbit (Dako, dilution 1:200) for 90 min at room temperature. After washing, the sections were incubated with avidin-biotin-horseradish peroxidase complex (Vectastain ABC Kit, Vecter laboratories, Newark, NJ, USA) for 45 min, with the detection of bound antibody performed with diaminobenzidine substrate. Slides were counter stained with haematoxylin and mounted in Aquatex (Merk, Darmstadt, Germany).

The 4-μm FFPE samples were placed in an oven for 2 h at 65 °C, and then deparaffinized in xylenes and rehydrated using an ethanol alcohol gradient followed by distilled water. The sections were immersed in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity at room temperature. After that, the sections were treated with Tris-EDTA Antigen Retrieval Solution (pH = 9.0) for 5 min in a pressure cooker for antigen retrieval. After the temperature of the antigen retrieval solution returned to room temperature, the sections were incubated with the mouse monoclonal anti-p53 antibody (diluted 1:50, Gene Tech Company Limited, Shanghai, China) at 37 °C for 1 h in a moist chamber and a biotinylated goat anti-mouse antibody (DAKO, Santa Clara, CA, USA) at 37 °C for 30 min on the next day. Subsequently, the sections were stained for protein detection in DAKO Liquid 3,′3-diaminobenzidine tetrahydrochloride (DAB) (DAKO) and counterstained with Mayer’s hematoxylin, then were dehydrated and mounted.