Biotin labeled secondary immunoglobulin

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).

The study was approved by the ethical committee of Sir Run Run Shaw Hospital, Zhejiang University School of Medicine, China. In addition, informed consent was obtained from all of the subjects involved. Human breast tumor tissue arrays were obtained from Sir Run Run Shaw Hospital. 314 female cases were recruited with ages ranging from 24 to 75. The IHC analysis staining of paraffin-embedded tissue sections was carried out using the Dako Envision system (Dako, Glostrup, Denmark) as previously described^{76 (link)}. 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 overnight at 4 °C. 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) at room temperature. Images were taken by using an Olympus camera and matched software. The following primary antibodies were used: rabbit-ASCT2 (Abcam, ab84903; diluted at 1:800), rabbit-SPOP (Affinit, DF12106; diluted at 1:800).

The antibody against Anillin was purchased from Abcam, USA. The Immunohistochemistry assay was carried out following our previously described methods 16 (link). In brief, the para-cancerous tissue sections with 4μm thickness were cut from paraffin-embedded tissue blocks, deparaffinized and rehydrated, and treated with 0.01mol/l citrate buffer (pH 6.0) for antigen retrieval. After blocking with goat serum solution for 45 min, the sections were incubated with primary antibodies at 4℃over-night. Antibodies used for IHC included antibodies against Anillin (1:200, Abcam, USA). After washing thrice with PBS, sections were incubated with biotin-labeled secondary immunoglobulin (1:100, DAKO, Glostrup, Denmark) for 1h at room temperature. The sections were then stained with diaminobenzidine (DAB, DAKO, Glostrup, Denmark) and were re-stained with hematoxylin at room temperature. Two experienced pathologists were assigned independently and blindly for detecting Anillin expression through IHC assay. The specimens were separated into two groups according to the staining intensity grade: no to low staining (0∼1+) and moderate to high staining (2+∼3+).