Immunohistochemical Analysis of Anillin Expression

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+).