Ab35219

The Modified Vaccinia Ankara (MVA) virus was kindly provided by Prof. Gerd Sutter (Ludwig-Maximilians-University of Munich, Germany). MVA was produced in BHK-21 cells and purified by ultracentrifugation through 36% sucrose in 1 mM Tris (pH 9.0) cushion and eluted in 1 mM Tris (pH 9.0). The virus titre for the MVA was determined by Immunocytochemistry (ICC) staining. Briefly, BHK-21 cells were infected with 10-fold dilutions of virus in MEM with 2.5% FBS for 24 hours after which the cells were fixed with methanol/acetone (1:1) solution at -20°C for 5 min. Infected cells were stained with rabbit anti-vaccinia antibody (ab35219, Abcam) followed by incubation with HRP-anti-rabbit secondary antibody (ab6721, Abcam). The virus-infected cells were visualized with DAB substrate (Sigma Aldrich). Western reserve strain of vaccinia virus (VACV) coding for murine DNA-dependent activator of IFN-regulatory factors (mDAI) [7 (link)] was produced in A549 cells and purified through 36% sucrose cushion ultracentrifugation and eluted in 1mM Tris (pH 9.0). The virus titer for VACV was determined by plaque assay [8 ].

For detection of virus encoded proteins, DT09/06 cells were harvested and resuspended in SDS sample buffer at 24, 48, 72, or 96 hours post virus infection (hpvi). DT09/06 tumors were harvested 28 days post infection (dpi) and shredded in SDS sample buffer using shredder tubes (Peqlab, Erlangen, Germany). Samples were separated by 10% SDS-Polyacrylamide gel electrophoresis and subsequently transferred onto a nitrocellulose membrane (Whatman GmbH, Dassel, Germany). After blocking in 5% skim milk in PBS, the membrane was incubated with rabbit anti-G6 antibody (affinity-purified polyclonal antibody raised against a GLAF-2 peptide, GenScript, Piscataway, NJ, USA) for detection of scAb GLAF-2 or polyclonal rabbit anti-vaccinia virus antibody (ab35219 Abcam, Cambridge, UK). The primary antibodies were detected using horseradish peroxidase-conjugated anti-rabbit (ab6721, Abcam, Cambridge, UK) secondary antibody, followed by enhanced chemiluminescence detection.

For histological studies of the xenograft model, tumors were excised and snap-frozen in liquid nitrogen. Tissue samples were sectioned (7-mm thickness) with the cryostat CM3050 S (Leica Microsystems GmbH, Wetzlar, Germany). The VACVs were labeled using CODEX tag-conjugated polyclonal rabbit anti-vaccinia virus (anti-VACV) antibody (ab35219 Abcam, Cambridge, UK) and ATTO 550 tag-conjugated fluorescent dye (kindly provided by G. Nolan, Stanford, CA, USA). The fluorescence-labeled preparations were examined using a BZ-X800 fluorescence microscope (Keyence, Osaka, Japan) equipped with the BZ-X800 Analyzer software (Keyence, Osaka, Japan).

We resuspended fixed cells from positive cultures in 100 µL of Permeabilization Medium (Invitrogen) with a rabbit polyclonal antibody vaccinia virus (Abcam, ab35219) at 1:2000 dilution (2 µg/mL) and incubated them for 20 min at room temperature in darkness. We removed the primary antibody by washing with blocking buffer (PBS, 5% FBS). Then we performed a secondary incubation with a polyclonal goat anti-rabbit IgG H&L Alexa Fluor® 488 antibody (Abcam, ab150077), which we added at 1:1000 dilution (2 µg/mL) and cells were incubated for 20 min at room temperature in darkness. After a PBS wash, we resuspended cells with 300 µL of PBS 1% PFA. To analyze the samples we used a FACSCalibur (Becton-Dickinson) and CellQuest and FlowJo v10.6.1 software to evaluate collected data.