Clone 5a1e

Explant cultures were embedded in paraffin and cut into 5 μM sections, after which hematoxylin and eosin (HE) staining was performed using standard methods. For immunohistochemistry, the sections were deparaffinized, blocked with 3% hydrogen peroxide and 10 mM citrate buffer, incubated with antibodies directed against Ki67 (Novocastra, NCL-L-Ki67-MM1) or cleaved caspase 3 (Cell Signaling Technologies, clone 5A1E, #9664) at 4 °C o/n, and with a biotinylated secondary antibody for 1 h at room temperature. Next, a chromogenic HRP-mediated diaminobenzidine staining method was used (Vectastain Elite ABC Kit, Vector Laboratories, USA), after which the samples were counterstained with Mayer’s hematoxylin. The stained sections were scanned using a digital slide scanner (Pannoramic 250 Slide Scanner, 3DHistech). Five representative images from cancerous areas were taken at 40x magnification. The number of stained cells/nuclei was quantified using thresholding within the Image J program, and the total number of tumor cells was calculated manually.

Mice were euthanized 72 hours following sham and tMCAO operation. Lung and spleen tissues were harvested, then fixed in 4% paraformaldehyde at 4°C overnight. After fixation, the tissues were embedded in tissue freezing medium, and sectioned to a thickness of 20 µm using cryostat. After 10 minutes incubation in 3% H₂O₂ (in methanol) at room temperature, the sections were incubated in the Tris‐buffered saline containing 0.3% Triton X 100% and 5% normal goat serum for 1 hour at room temperature, then incubated with primary antibody that recognizes the cleaved (Asp175) form of caspase 3 in a dilution of 1:500 (clone 5A1E, Cell Signaling Technology, Danvers, MA) overnight at 4°C. The sections were washed, then incubated with the SignalStain Boost IHC detection reagents (Cell Signaling Technology) for 30 minutes at room temperature. The horseradish peroxidase activity was detected with SignalStain DAB substrate kit (Cell Signaling Technology). The sections were counterstained with hematoxylin, dehydrated, and mounted. Images were collected with an Olympus Slide Scanner at 10x magnification.

The different antibodies used were mouse monoclonal anti-K8 antibody (clone M20, Sigma-Aldrich, Darmstadt, Germany), mouse monoclonal anti-C terminal K8 antibody (clone 1E8, Abcam, Cambridge, UK), rabbit polyclonal anti-C terminal K8 antibody (SAB4501654, Sigma-Aldrich, Darmstadt, Germany), rabbit polyclonal anti-N terminal K8 antibody (ab137855, Abcam, Cambridge, UK), proprietary mouse monoclonal anti-K8 antibodies (clone D-A10 and clone D-D6), mouse monoclonal anti-uPA antibody (ab131433, Abcam, Cambridge, UK), mouse monoclonal anti-plasminogen antibody (ab38157 Abcam, Cambridge, UK), rabbit polyclonal anti-caspase 3 cleaved (Asp 175) antibody for IF analysis (9661, Cell Signaling Technology, Leiden, The Netherlands), rabbit monoclonal anti-cleaved caspase 3 antibody for IHC analysis (clone 5A1E, Cell Signaling Technology, Leiden, The Netherlands), mouse monoclonal anti-caspase 3 and cleaved caspase 3 Alexis antibody for Western blot (WB) analysis (Enzo Life Science, Farmingdale, NY, USA), mouse monoclonal anti-Ku80 antibody (ab3715, Abcam, Cambridge, UK), mouse monoclonal anti-histone H3 antibody (ab1791, Abcam, Cambridge, UK), anti-Na+/K+ ATPase alpha (H3) (SC-48345, Santa Cruz Biotechnology, Santa Cruz, CA, USA) rabbit polyclonal anti-GFP antibody (ab290, Abcam, Cambridge, UK), and mouse monoclonal anti-Ki67 antibody (Clone Mib1, M7240 DAKO, Santa Clara, CA, USA).