Ab6526

Tumor tissues were fixed in 10% formalinand subsequently processed with an automated tissue processor (Leica ASP 6025) and paraffin embedded. Sections (4 µm-thick) were mounted on coated glass slides, and heated at 60 °C for 60 min. One section was stained with hematoxylin and eosin for light microscopy analysis, while other sections were prepared for immunohistochemical analyses for which standard protocol was applied [49 (link)]. Tissue sections for immunohistochemical staining was performed with an automated immunostainer (Bond RX Max, Leica Biosystems, Buccinasco, Milan, Italy) using anti-CD 31 (ready to use, clone JC70A, Dako Agilent, Santa Clara, CA, USA) and anti-Ki67 (1:500) (ab6526, Abcam, Cambridge, UK) antibodies. A direct count of positive cells was conducted on ten fields for all the cases. The sections were stained with DAB and then slightly counterstained with Mayer hematoxylin, to be analyzed using a microscope (Leica LDM108) and digital image-capture computer system. Two senior pathologists (ChM and SC) analyzed the immunohistochemical results independently, blind to treatment, discriminating positively (brown color) and negatively (blue color) stained cells through manual counting. The results are expressed as percentage of positive cells.

Paraffin-embedded samples of primary carcinomas were immunostained with primary rabbit anti-human SATB-1 (1:100, ab92307, Abcam), rabbit anti-human α-SMA (1:100, ab32575, Abcam), and rabbit anti-human SDF-1 (1:500, ab9797, Abcam) and Ki-67 (1:500, ab6526, Abcam) antibodies. The expression levels of SATB-1 and SDF-1 were scored semi-quantitatively, based on staining intensity and distribution, using the immunoreactive score as described elsewhere. Staining was assessed by two pathologists under double-blind conditions according to the scoring criteria. These procedures are described in the Supplementary Methods.

For Western blotting, 10⁴ cells/µl were used per lane. Immunodetection was performed using 5%-powdered milk in PBS-T (1xPBS, pH 7.4 and 1% Tween20) for blocking (Roth, Germany). Primary antibodies, anti-p21 mouse antibody (OP64; Calbiochem; dilution 1∶200), anti-p16 mouse antibody (550834; BD Pharmingen; 1∶200), anti-p27 rabbit antibody (sc-528; Santa Cruz; 1∶200), anti-γH2AX (07-164; Millipore; 1∶50), anti-Cyclin D1 rabbit antibody (ab16663; Abcam; 1∶500), anti-Cyclin D2 mouse antibody (ab3805; Abcam; 1∶500), anti-Ki-67 mouse antibody (ab6526; Abcam; 1∶200), anti-HES1 rabbit antibody (sc-25392; Santa Cruz; 1∶200), anti-Bcl-2 (IMG-80093; IMGENEX; 1∶200), anti-Bax (IMG-80165; 1∶250) and anti-tubulin mouse antibody (T-9026; SIGMA-Aldrich; 1∶5000) were diluted as indicated in 5%-powdered milk (in PBS-T) and incubated for one hour at room temperature. Washing steps were performed 3×10 min in 1×PBS-T. The secondary horseradish peroxidase-labeled antibodies (Jackson Immuno Research Lab) were incubated for 1 hr at room temperature. Detection of horseradish peroxidase was performed using ECL-detection system and radiographic film (GE Healthcare, Germany). After film development, quantification of signal intensities of the bands in the Western blots was carried out using Metamorph software.