Vector novared

A GBM tissue microarray was constructed consisting of three representative 1-mm cores from formalin-fixed, paraffin-embedded (FFPE) tissue blocks from each one of 50 GBM patients. All GBM tumor specimens were obtained from patients who underwent surgery at Ronald Reagan UCLA Medical Center. All samples collected were under patients’ written consent, and were approved by the UCLA Institutional Review Board. Twenty-eight were newly diagnosed GBM and 22 were recurrent GBM. 21 of the newly diagnosed GBM were primary GBM and 7 were secondary GBM. Eighteen recurrent GBM were primary GBM and 4 were secondary GBM. Immunohistochemical analysis was performed on 5-μm FFPE sections of TMAs. Antigen retrieval was performed by immersing TMA slides into a 1X Dako Target Retrieval buffer for 40 min in a pressure cooker. Endogenous peroxidase activity was quenched with 3% hydrogen peroxide in PBS. TMA sections were incubated with diluted primary antibodies ribosomal protein S11 (RPS11) at 1:25, ribosomal protein S20 (RPS20) at 1:100, Vascular endothelial growth factor A (VEGFA) at 1:50 (Prestige Antibodies®, Sigma, St. Louis, MO) for 16 h at 4°C, followed by incubation with MACH-3 (Rabbit) or MACH-4 (Mouse) secondary antibodies (Biocare Medical, Concord, CA) for 30 minutes at room temperature. Subsequent immunodetection was completed using Vector NovaRed (Vector Laboratories, Burlingame, CA).

Patient tumor material or xenografts were fixed in 4% formalin prior to paraffin embedding. Sections of 5 μm were prepared on a microtome. Tissue sections were deparaffinized and heat mediated antigen retrieval was performed using Tris-EDTA buffer solution pH 9 for Hedgehog staining or 10 mM sodium citrate solution pH 6 for other stainings. Endogenous peroxidase activity was blocked with 3% hydrogen peroxide in PBS. Aspecific staining was blocked using Ultra-V Block (Immunologic) for 10 min at room temperature. Primary antibodies were diluted in normal antibody diluent (KliniPath), applied on tissue sections and incubated overnight at 4°C in a humidified chamber. For amplification of signal Brightvision + post antibody block (Immunologic) was used prior to the addition of the secondary antibody; poly-HRP-anti Ms/Rt/Rb IgG (Immunologic) both for 30 min at room temperature. Visualization was performed using Vector® NovaRED™ (Vector Labs) according to manufacturer’s protocol, counterstained with 30% haematoxylin and tissue sections were mounted with non-aqueous medium. Antibodies used for immunohistochemistry were: anti-alpha smooth muscle actin (Abcam, 1:1000); anti-Hedgehog (clone H160, Santa Cruz, 1:1500), anti-Cytokeratin 19 (clone RCK108, BioGenex, 1:1000), anti-pan cytokeratin (clone AE1/AE3, Dako, 1:100).

To investigate the production of organic matrix by cells grown on scaffolds, an immunohistochemical staining for Type I Collagen (COL1A1) (MILLIPORE, Temecula, CA, USA) was performed. After endogenous peroxidase blocking and antigen unmasking, sections were incubated with primary mouse monoclonal anti-human antibodies overnight at 4 °C. Secondary antibodies (Vectastain Universal Quick kit; Vector Laboratories, Burlingame, CA, USA) and a development kit (Vector Nova Red; Vector Laboratories) were applied following the manufacturer’s instructions. Representative images were captured with an optical microscope (BX51, Olympus Italia) connected to an XC50 Olympus digital camera (Olympus Italia) and to an image analyzer system (CellSens Dimension, Life Sciences Imaging Software, Olympus Italia).

Human and mouse liver specimens were fixed overnight in 4% paraformaldehyde and embedded in paraffin. Tumors arising in mice from different oncogene combinations and used for Fascin1 IHC were induced in FVB/N females (see references in the pertaining section of text). Sections were done at 5 μm in thickness. Liver lesions were evaluated and classified by two board-certified pathologists and liver experts (S.R. and M.E.). For immunohistochemistry, slides were deparaffinized in xylene, rehydrated through a graded alcohol series, and rinsed in PBS. Antigen unmasking was achieved by boiling in 10 mM sodium citrate buffer (pH 6.0) for 10 min, followed by a 20-min cool down at room temperature. After a blocking step with the 5% goat serum and Avidin-Biotin blocking kit (Vector Laboratories, Burlingame, CA), human tissue slides were incubated with primary antibody overnight at 4 °C (see Table 2). Slides were subjected to 3% hydrogen peroxide for 10 min to quench endogenous peroxidase activity and, subsequently, the biotin conjugated secondary antibody was applied at a 1:500 dilution for 30 min at room temperature. Immunoreactivity was visualized with the Vectastain Elite ABC kit (Vector Laboratories, Burlingame, CA), using Vector NovaRed (Vector Laboratories) as the chromogen. Slides were counterstained with hematoxylin.

Paraffin embedded formalin-fixed sections (4 μm) of non-affected liver tissue from control patients (n = 3) were stained using sequential immunohistochemistry^{44 (link)}. Sections were deparaffinized and rehydrated in up to 96% ethanol. Endogenous peroxidase activity was blocked by incubation with 0.3% H2O2 in methanol. Antigen retrieval was performed at 120 °C under pressure in a Tris/EDTA buffer. The sections were stained overnight with anti-CD69 (mouse IgG1, clone CH11, Abcam, Cambridge, UK), followed by incubation with polyHRP-anti-mouse IgG (ImmunoLogic, Duiven, Netherlands). HRP activity was demonstrated with VectorNovaRed (Vector Laboratories, CA, USA). The slides were counterstained with hematoxilin, coverslipped and scanned in a Philips Ultra Fast Scanner 1.6RA (Philips, Eindhoven, Netherlands). After removal of the coverslip, the sections were stripped in a Tris-SDS buffer at 50 °C. Next, they were incubated with anti-CD103 (rabbit IgG, clone EPR4166, Abcam, Cambridge, UK) and subsequent polyHRP-anti-rabbit IgG (ImmunoLogic, Duiven, Netherlands). HRP activity was demonstrated as above. This procedure was repeated with anti-CD8 (mouse IgG1, clone C3/144B, Dako, Glostrup, Denmark) and anti-CD3 (rabbit IgG1, clone Sp7, Thermo scientific, NH, USA). Images were analysed with Fiji Image J software^{45 (link)}. Pseudocolors were applied to provide co-localization images.