Immunohistochemical Staining of Paraffin Sections

Immunohistochemical analysis staining of paraffin‐embedded tissue sections was carried out using the Dako Envision System (Dako, Glostrup, Denmark) following the manufacturer's protocols. Briefly, the sections were submerged in boiling 10 mmol/L sodium citrate (pH, 6.0) for 2 min in a pressure cooker. After being treated with 0.3% hydrogen peroxide for 10 min to block endogenous peroxidase, the sections were incubated with primary antibody for 1 h at room temperature. After washing, the sections were incubated with biotin‐labeled secondary immunoglobulin (Dako) for 40 min at room temperature, followed by incubation with 3,3′‐diaminobenzidine (Dako), also at room temperature. The primary antibodies used were anti‐fascin‐1 mouse monoclonal antibody (clone 55k‐2; diluted at 1:100; Santa Cruz Biotechnology, Santa Cruz, CA), ready‐to‐use anti‐ER rabbit monoclonal antibody (clone SP1, Dako), ready‐to‐use anti‐PR mouse monoclonal antibody (clone PgR636, Dako), HercepTest (Dako), and ready‐to‐use anti‐Ki‐67 mouse monoclonal antibody (clone MIB‐1, Dako).