M0851

Primary antibodies used were as follows: mouse anti-SSEA-4 (1:100, MAB4304, Millipore), goat anti-Nanog (1:20, AF1997, R&D Systems), rabbit anti-Oct4 (1:200, ab19857, Abcam), mouse anti-α-SMA (1:200, M0851, Dako), goat anti-Sox17 (1:200, AF1924, R&D Systems), rabbit anti-Tuj1 (1:2000, MRB-435P, Covance), mouse anti-Flag M2 (ICC, 1:500; WB, 1:100, F1804, Sigma), mouse anti-HA Tag (6E2) (ICC, 1:100; WB, 1:1000, #2367, Cell Signaling), rabbit anti-Tom20 (1:100, sc-11415, Santa Cruz), and mouse anti-GAPDH (1:1000, NB600-502, Novusbio). Alexa Fluor 488 (A11055 or A11034, Molecular Probes), Alexa Fluor 594 (A11005, Molecular Probes) and Alexa Fluor 647 (ab150075, Abcam)-conjugated secondary antibodies were used for immunofluorescent study.

Reagents were as follows: human plasma fibronectin (20 μg/mL, F1056; Sigma-Aldrich, Munich, Germany), human recombinant TGFβ1 (10 ng/mL, 100-21C; Peprotech, London, UK), αII-spectrin siRNA (25 ng/cm², EHU093741; Sigma-Aldrich), βII-spectrin siRNA (25 ng/cm², EHU081451; Sigma-Aldrich), Renilla luciferase siRNA (25 ng/cm², EHURLUC; Sigma-Aldrich), Alexa647-labeled streptavidin (8 μg/mL, S32357; Thermo Fisher Scientific, Landsmeer, The Netherlands), TRITC labeled-Phalloidin (100 nM, P1951; Sigma-Aldrich). Antibodies used: mouse anti-αII-spectrin (2 μg/mL, sc-376849; Santa Cruz, Dallas, USA), mouse anti-βII-spectrin (2 μg/mL, sc-376487; Santa Cruz), mouse anti-αSMA (0.28 μg/mL, M0851; DAKO; Glostrup, Denmark), mouse anti-collagen type I (1 μg/mL, ab90395; Abcam, Cambridge, UK), mouse anti-vinculin (9.3 μg/mL, V9131; Sigma-Aldrich).

Following tumor resections, 2 mm thick sections perpendicular to the injection columns, were fixed in 10% buffered formalin for 48 hours, scanned and processed as previously described [5 (link)]. Rabbit anti-CC3 antibody (Cell Signaling, 1:150 dilution), mouse anti pHH3 (Cell Signaling, 1:200 dilution), mouse anti-GLUT1 (Abcam ab40084, 1:250 dilution), rabbit anti-CD31 (Abcam ab28364, 1:250 dilution), rabbit anti-SMA (Dako M0851, 1:1000 dilution), rabbit anti-S100A9 (Abcam 63818, 1:5000 dilution) were used for IHC analysis. For immunofluorescent detection, secondary antibodies conjugated to AlexaFluor647 (Jackson Immunoresearch, 1:600 dilution) and AlexaFluor555 (Invitrogen, 1:500 dilution) was applied according to manufacturer’s instructions and tissues counterstained with DAPI.

Immunohistochemistry was performed as previously described (Warnock et al., 2012 (link)) for type I collagen (AB749P; 1:100 dilution; Millipore), type II collagen (AB746P; 1:100: Millipore), macrophage MAC387 receptor to determine type A synoviocyte content, (CBL260; 1:200 dilution; Millipore); and alpha smooth muscle actin (M0851; 1:30; Dako). Extracellular and intracellular immunoreactivity intensity and prevalence was scored as previously described (Wakshlag et al., 2011 (link)) with some modifications: immunoreactivity was localized to intracellular or extracellular staining, and ECM immunoreactivity intensity was described and scored, as negative (0), mild (1), moderate (2), or strong (3) staining. As determined by hand count, intracellular immunoreactivity and extracellular immunoreactivity was categorized as positive in <10%, 10–50%, or >50% of cells and sample area, respectively. Each of these histologic observations was assigned a score (Table 2). Then a histologic intensity coefficient was calculated for each ECM component, as follows: [[(Extracellular matrix staining intensity score) × (percentage area coverage of positive staining score)] + [(Intracellular staining intensity score) × (percentage positive staining cells score)]]/2 (Table 1).

Passage 1 EC and SMC were grown to confluence on plastic dishes or on chitosan/PCL membranes. After that, they were fixed with 4% PFA (paraformaldehyde) for 10 min, permeabilized with 0.05% Triton X-100 for 10 min, and blocked with 1% BSA (bovine serum albumin). The cells were stained with primary antibodies overnight at 4 °C, washed with PBS and incubated with secondary antibodies for 1 h at room temperature. The stained cells were analysed with an inverted fluorescence microscope (Nikon Ti-E) using Nikon AR software.
The following primary antibodies were used: anti-human CD31 (M0823, DAKO, 1:50), anti-α-SMA (DAKO, M0851, 1:50), anti-smooth muscle myosin heavy chain 11 (Abcam, ab82541, 1:500), anti-human CD90 (eBioscience, 14090982, 1:100), anti-Von Willebrand factor (Abcam, ab6994, 1:200), anti-fibronectin (Abcam, ab6328, 1:200), anti-elastin (Abcam, ab21610, 1:200), and anti-collagen IV (Life Span, 1:200).
The following secondary antibodies were used: Alexa Fluor 568 goat anti-mouse IgG1 (Life Technologies, A21124, 1:400), Alexa Fluor 488 goat anti-mouse IgG1 (Life Technologies, A21121, 1:400), Alexa Fluor 568 goat anti-mouse IgG2a (Life Technologies, A21134, 1:400), Alexa Fluor 568 goat anti-mouse IgG (H + L) (Life Technologies, A11031, 1:400), and Alexa Fluor 488 goat anti-mouse IgG (H + L) (Life Technologies, A11029, 1:400).

Extracted tumours were fixed in 10% buffered formalin (Sigma) for 24-48 h at room temperature and then stored in 70% ethanol at 4°C. Samples were then paraffin-embedded, sectioned at 4 μm thickness and stained with haematoxylin and eosin (H&E). Ki67 (NB110-90592, Novus), TUNEL (homemade by the Pathology Research Program Laboratory), ER (ab80922, Abcam), PR (NCL-PR-312, Leica), HER2 (RM9103, Thermo Fisher Scientific), CK5 (NCL-LCK5, Leica), EGFR (28-0005, Invitrogen), p63 (VP-P960, Vector) and SMA (M0851, Dako) were used to further analyse tumour sections. All immunohistochemistry was performed as a service by the Pathology Research Program Laboratory (University Health Network, Toronto, Canada).