Ab1056

Tumors were harvested one week after the fourth treatment, embedded in optimal cutting temperature compound (O.C.T.) (Sakura Finetek, Torrance, CA), and stored at −20 °C. The sections (8 μm) were subjected to either hematoxylin-eosin (H&E) or immunohistochemical staining (IHC) as described previously [48 ]. For IHC staining, the sections were incubated with goat-anti-Ad polyclonal antibody (AB1056, Millipore, Billerica, MA) and diluted (1:800) for 1 h at room temperature. The signals were amplified by a biotinylated anti-goat IgG diluted (1:200) in conjunction with VECTASTAIN avidin-biotin complex method kit (Vector Laboratories, Burlingame, CA). Visualization was achieved using 3,3-diaminobenzidine tetrahydrochloride (ImmPACT DAB peroxidase substrate, Vector Laboratories). Hematoxylin was used as a counterstain. Images were acquired at X200 magnification by using an Olympus BX53 microscope (Olympus, Center Valley, PA).

Paraffin-embedded sections of mice brains were immunostained for antibodies specific to adenoviral mouse-hexon (1:1000; AB1056, Millipore), adenovirus rabbit-E1A, (1:1000; Santa Cruz Bio-technology, Santa Cruz, CA), CD3 (1:300; clon SP7, NeoMarkers, Fremont, CA), CD4 (1:1000; EPR19514, ab183685 Abcam, Cambridge, MA) CD8a (1:1000, (D4W2Z) #98941 Cell Signaling, Danvers, MA) and FoxP3 (1:400; clon JFK-16s, ref. 14–5773 eBiosciences, Thermo Fisher, Waltham, MA) following conventional procedures. For the immunohistochemical staining, Vectastain ABC kits (Vector Laboratories Inc., Burlingame, CA) were used according to the manufacturer’s instructions.