Ki 67

All of the fixed samples were dehydrated in a graded alcohol series. Tissue samples were eventually embedded in paraffin before being sectioned. The rehydrated sections were employed for immunostaining. Primary antibodies specific to Ki67 (Epitomics, Burlingame, CA, USA), PH3 (Cell Signaling Technology, USA) and PCNA (Cell Signaling Technology, Beverly, MA, USA) were used. Sections incubated without primary antibodies were used as negative controls. High-temperature heating was used for antigen retrieval. A Histostain-SP Kit (Zhongshan Golden Bridge Biotechnology, Beijing, China) was applied to visualize the antigen. For immunofluorescence, liver sections (8 μm) were fixed in 4% paraformaldehyde solution. Primary antibodies specific to p27 (ABclonal Technology, Wuhan, China) were used, and the sections were stained with Cy3-labeled secondary antibodies (Jackson ImmunoResearch Laboratories, West Grove, PA, USA) before being counterstained with 4′,6-diamidino-2-phenylindole. Immunofluorescence images were captured via a Leica TCS SP5 confocal scanning laser microscope (Leica, Solms, Germany).

Immunohistochemistry was performed in 5-μm-thick paraffin-embedded sections using specific antibodies as indicated. Tissues were fixed in 10% neutral buffered formalin. Microwave heating was used for antigen retrieval. For immunohistochemistry analysis, antibodies specific to ERα (Dako, 1:100), p-ERα (Santa Cruz Biotechnology, 1:100), progesterone receptor (PR) (DAKO, 1:100), Ki-67 (Epitomics, 1:200), COX2 (Santa Cruz Biotechnology, 1:200), CD31 (BD, 1:50) were used in 5-μm thick paraffin-embedded sections. A Histostain-SP Kit (Zhongshan Golden Bridge Biotechnology) was used to visualize the antigen. For immunofluorescence, double-immunofluorescence staining for SMA (Bio Genex, 1:100) and cytokeratin (DAKO, 1:100) was performed in paraffin-fixed sections and secondary antibodies conjugated with Cy2 and Cy3 dyes were used, respectively. Immunofluorescence for Laminin (Sigma, 1:200) and cytokeratin was performed in fresh-frozen sections. Nuclear staining was performed using Hochest 33343 (Sigma, 0.1 μg/ml). All frozen sections were fixed in 10% neutral buffered formalin and incubated with primary antibody at 4 °C overnight, followed by incubation with secondary antibody at room temperature for 1 h. Immunofluorescence images were captured in a Zeiss LSM 510 confocal scanning laser microscope.