Cryostat 2800 frigocut

For immune-histochemistry, tumors were excised and snap-frozen in liquid N₂, followed by fixation in 4% paraformaldehyde/PBS pH 7.4 for 16 h at 4°C. Fixed tumors were dehydrated in 10% Sucrose/PBS for 3–4 h followed by 30% sucrose/PBS for 12 h and finally embedded in Tissue-Tek® O.C.T. (Sakura Finetek Europe B.V., Alphen aan den Rijn, Netherlands). Tumor samples were sectioned (15 μm) with the cryostat 2800 Frigocut (Leica Microsystems GmbH, Wetzlar, Germany) and stored at -80°C. Antibody-labeling was performed following fixation in ice-cold aceton. Endothelial cells were labelled with the hamster anti-mouse CD31 antibody (Chemicon, International, Temecula, CA) and Cy3-conjugated secondary donkey anti-hamster antibody obtained from Jackson ImmunoResearch (West Grove, PA). The primary antibody was incubated for 1 h. After washing with PBS, sections were labeled for 30 min with the secondary antibody and finally mounted in Mowiol 4–88.

For histological studies, tumors were excised and snap-frozen in liquid nitrogen, followed by fixation in 4% paraformaldehyde/PBS at pH 7.4 for 16 h at 4 °C. After dehydration in 10% and 30% sucrose (Carl Roth, Karlsruhe, Germany) specimens were embedded in Tissue-Tek O.C.T. (Sakura Finetek Europe B.V., Alphen aan den Rijn, The Netherlands). Tissue samples were sectioned (10 mm thickness) with the cryostat 2800 Frigocut (Leica Microsystems GmbH, Wetzlar, Germany). Labeling of tissue sections was performed as described in detail elsewhere [25 (link)]. In this case, VACVs were labeled using polyclonal rabbit anti-vaccinia virus (anti-VACV) antibody (Abcam, Cambridge, UK), which was stained using Cy2-conjugated donkey anti-rabbit secondary antibodies obtained from dianova (Hamburg, Germany). Endothelial blood vessel cells were stained with a hamster monoclonal anti-CD31 antibody (Merck Millipore KGaA, Darmstadt, Germany, MAB1398Z) and a DyLight649-conjugated goat anti hamster secondary antibody from dianova.
The fluorescence-labeled preparations were examined using a TCS SP2 AOBS confocal laser microscope (Leica Microsystems GmbH, Wetzlar, Germany) equipped with the LCS 2.16 software (102461024 pixel RGB-color images). Digital images were processed with Photoshop 7.0 (Adobe Systems, Mountain View, CA, USA).