Ki 67 antibody

Prostate glands were obtained from 20-week-old mice. The prostates were fixed in 10% neutral buffered formalin and embedded in paraffin blocks. Serial sections (5 μm) were cut and submitted to routine hematoxylin and eosin (H&E) staining. Immunostaining with Ki-67antibody (12202; Cell Signaling Technology, Danvers, MA, USA) and p21 antibody (187; Santa Cruz Biotechnology, Dallas, TX, USA) was performed at Center for Anatomical, Pathological and Forensic Medical Researches at Kyoto University. Ki-67-positive epithelial cells were counted in each lobe (VP, LP and DP).

To determine the changes in cytoskeleton structure and expression of different markers, CiSCs, BM-MSCs, and MCF7 cells were seeded on glass slides precoated with poly-d-lysine (Sigma-Aldrich, USA). Cells were fixed in 4% paraformaldehyde, permeabilized with 0.1% Triton X-100, and blocked with 4% BSA. Cells were then stained with Alexa Fluor® 488 Phalloidin (Molecular Probes, USA), α-tubulin antibody (Cell Signaling Technology, USA), Ki-67 antibody (Cell Signaling Technology, USA), Oct-4 antibody (Cell Signaling Technology, USA), Sox2 antibody (R&D Systems, USA), Nanog antibody (Bioss Antibodies, USA), E-Cadherin antibody (Cell Signaling Technology, USA), N-Cadherin antibody (Abcam, USA), Snail + Slug antibody (Abcam, USA), ALDH1A1 antibody (Pierce Antibodies, USA), and β-Catenin antibody (Cell Signaling Technology, USA). Cells were labeled with the appropriate Alexa Fluor® secondary antibodies (Molecular Probes, USA) and counterstained with Hoechst 33342 (Molecular Probes, USA) to visualize the cell nucleus. Cells were imaged either under a 60× or 100× objective with a Nikon A1R inverted laser scanning confocal microscope (Nikon microsystems, France).

Lentivirus-stabilized KGN cell lines, which stably overexpressing lnc-GULP1–2:1 (Lv-lnc-GULP1–2:1) and its control (Lv-EGFP), were re-inoculated on glass coverslips pre-coated with poly-lysine and cultured for 24 h in an atmosphere of 5% CO₂ at high humidity. Cells were treated with 4% paraformaldehyde for 20 min, then gently washed with phosphate buffered saline (PBS) for 3 × 3 min. Next, cells were permeabilized with 0.5% Triton X-100 in PBS for 20 min and blocked by using 5% bovine serum albumin (BSA) in TBST for 30 min at room temperature. After blocking, cells were incubated with COL3A1 antibody (1:100, Santa Cruz) or Ki-67 antibody (1:1000, Cell signaling technology, Beverly, USA) at 4 °C overnight; PBS was used as a negative control. Then cells were washed 3 × 3 min with PBS at room temperature and incubated with Alexa Fluor 594-AffiniPure Goat Anti-Rabbit IgG (1:600, Jackson, Pennsylvania, USA) for 30 min. After washing with PBS, the nuclei were stained with 4′, 6-diamidino-2-phenylindole (DAPI) (5 μg/ml). Then the coverslips were washed again with PBS 4 × 5 min. Finally, the coverslips were mounted with anti-fade Mounting Medium (Beyotime Biotechnology, Shanghai, China), and the image was observed and collected under a laser scanning microscope (Carl Zeiss, Oberkochen, Germany).