Villin Monoclonal antibody

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Tissue microarrays (TMAs) were constructed as previously described [20-22]. 4 μm-thick tissue microarray sections were cut from the TMA blocks, dewaxed in xylene, rehydrated in alcohol, and incubated in 3% hydrogen peroxide for 10 min to block endogenous peroxidase activity. Antigen retrieval was performed by microwave oven heating (10 min) in 0.01 M sodium citrate buffer (pH 6.0). Sections were incubated with 10% normal goat serum in PBS for 15 min at room temperature to block nonspecific binding. Then sections were incubated overnight at 4°C with each of the four primary antibodies: rabbit anti-human tensin polyclonal antibody (sc-28542, 1:500 dilution, Santa Cruz Biotechnology, Inc. Dallas, Texas, USA), rabbit anti-human profilin-1 monoclonal antibody (EPR6304, 1:1000 dilution, Epitomics, Burlingame, California, USA), mouse anti-human villin-1 monoclonal antibody (66096-1-lg, 1:500 dilution, Proteintech Company, Wuhan, Hubei, China) and mouse anti-human Talin monoclonal antibody (sc-365460, 1:200 dilution, Santa Cruz Biotechnology, Inc. Dallas, Texas, USA). Then, bound antibodies were visualized with a PV-9000 2-Step Plus Poly-HRP Anti-Mouse/Rabbit IgG Detection System (ZSGB-BIO, Beijing, China) and a Liquid DAB Substrate Kit (Invitrogen, Carlsbad, CA, USA). Negative controls were prepared by substituting normal mouse or rabbit IgG for the primary antibodies.

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