Rabbit anti vaccinia virus antibody

Tumors harvested at the time of sacrifice were fixed with 10% formalin. Subsequently, tumors were paraffin-embedded and 5 μm thick sections were obtained. The slides were deparaffinized followed by heat-mediated antigen-retrieval per manufacturer’s protocol (IHC World, Ellicott City, MD). Tumor sections were then permeabilized with methanol and blocked for 30 minutes with TNB Blocking buffer (PerkinElmer, Waltham, MA). Tumor sections were washed and incubated overnight in a humidified chamber at 4°C with a rabbit anti-vaccinia virus antibody (Abcam, Cambridge, MA, RRID:AB_778768) 1:100 in TNB Blocking buffer. The next day, tumor sections were stained with Alexa Fluor-488-conjugated goat anti-rabbit (Abcam, Cambridge, MA, RRID:AB_2630356) for 1 hour at room temperature. Finally, the sections were counterstained with 4′6-diamidino-2-phenylindole (DAPI). IHC for CD8 and Granzyme B was performed by the Pathology Core at City of Hope. Images were obtained using the Nanozoomer 2.0HT digital slide scanner (Hamamatsu Photonics, Hamamatsu City, Shizuoka Pref., Japan) or Ventana iScan HT (Roche, Basel, Switzerland).

Tumor tissues were harvested and fixed for 48 – 72 hours in 4% paraformaldehyde (4%PFA, Boston BioProducts) and stored in 70% ethanol until further processing. Paraffin-embedded tumor sections (10 μm) were deparaffinized followed by heat-mediated antigen retrieval for 30 minutes in IHC-TEK epitope retrieval solution (IHC World). Following antigen retrieval, tumor sections were permeabilized with 100% methanol. Sections were blocked for 30 minutes with Tris-NaCl (TNB) blocking buffer (PerkinElmer), and then incubated with rabbit anti-vaccinia virus antibody (Abcam) diluted 1:100 in TNB blocking buffer overnight in a humidified chamber at 4°C. Following incubation, tumor sections were wash and incubated with Alexa-Fluor-488-conjgated goat anti-rabbit secondary antibody (Abcam) for 1 hour at room temperature. Finally, the sections were counterstained with DAPI.
Immunohistochemistry for CD3 and CD8 were performed by the Pathology Core at City of Hope. Briefly, deparaffinized tumor sections (10 μm) were stained with hematoxylin & eosin (H&E, Sigma-Aldrich), rat anti-mouse CD3 (Abcam), and rat anti-mouse CD8a (Novus Biologicals). Images were obtained using the Nanozoomer 2.0HT digital slide scanner and the associated NDP.view2 software (Hamamatzu). For CD8 quantification after IHC staining, ImageJ (NIH) analysis was performed as per the standard recommended algorithm^{55 (link)}.