Powerdab

Proliferation and apoptosis in the tumor nodules and organs were detected with IHC stainings for Ki-67 and cleaved caspase-3, respectively. Sections of 4 µm were cut, deparaffinized and heat-induced antigen retrieval was performed with 1x Sodium Citrate pH 6 at 98°C, followed by peroxidase blocking. Slides were incubated with the primary antibody Recombinant Anti-Ki-67 antibody 1:200 (anti-rabbit, Abcam) or cleaved caspase-3 1:200 (anti-rabbit, Cell Signaling) overnight at room temperature. The next day, sections were incubated with the secondary antibody Poly-HRP-anti-Rabbit IgG (Immunologic) for 60 minutes at room temperature. Afterwards, the sections were stained with PowerDAB (Immunologic), counterstained with hematoxylin (Fluka) and mounted with Pertex. Representative pictures were taken with a light microscope (Olympus) at a magnification of 40x. Percentage positive staining was analyzed using the software program QuPath version 0.2.3.

Immunohistochemistry on formalin-fixed, paraffin-embedded (FFPE) tissue sections was performed as previously described using antibodies against MET and P-MET (clone D1C2 and D26, respectively, both CST) [42 (link)]. Antibodies were visualized via sequential incubations with biotinylated secondary antibodies, avidin–biotin complexes (Vector laboratories, Burlingame, CA, USA) and 3,3′-diaminobenzidine solution (Power-DAB, ImmunoLogic, Duiven, The Netherlands).

Detection and scoring of immunofluorescence γ-H2AX and Rad51 in cell lines, was performed as previously described [15 (link), 27 (link)]. Xenografts were fixated in 3.6% paraformaldehyde (Aurion) and embedded in paraffin. Sections of 4 μm were prepared for detection of both cleaved-Caspase3 and γ-H2AX and heat-induced antigen retrieval was performed at pH 6. CASP3 sections continued with peroxidase blocking for 20 min and serum blocking using Ultra-V (Immunologic) for 5 min. Primary antibody Cleaved-Caspase3 (anti rabbit, Cell Signaling) was applied 1:200 overnight at 4°C. Thereafter, sections were incubated with Powervision Poly-HRP-GAM/R/R IgG (Immunologic) for 30 min and PowerDAB (Immunologic) for 1–2 min, counterstained with haematoxylin (Fluka) and mounted with pertex.
For γ-H2AX, sections were blocked after antigen retrieval in PBTB: PBS containing 0.1% Tween20 and 2% Bovine Serum Albumin (BSA) and incubated with primary antibody mouse monoclonal anti- γ-H2AX (Millipore) for 90 min (1:100 in PBTB) at room temperature. Secondary antibody goat anti-mouse Cy3 (Jackson Immunoresearch) was applied for 60 min, and DAPI (Sigma-Aldrich) was used as counterstain. After washing, sections were embedded in Vectashield and analysed by microscopy.