Anti ad2 5 e1a antibody

To detect FAP and E1A-Adenovirus expression in tumors, immunohistochemistry (IHC) was performed using OCT-embedded sections (5 μm thick) of freshly frozen tumor tissues. Sections were fixed with 2% of PFA at room temperature and endogenous peroxidases were blocked by incubation in 3% H₂O₂. Next, sections were blocked for 1 h with 10% of normal goat serum diluted in 1% BSA, PBS-Tween. For FAP detection, primary antibody incubation was performed overnight at 4 °C using a biotinylated polyclonal sheep anti-human/mouse FAP antibody (5 μg/ml) or its isotype sheep IgG (R&D systems) in 5% of goat serum. For adenovirus detection, the primary antibody used was an anti-Ad2/5 E1A antibody (Santa Cruz Biotechnology) diluted 1/200 in PBS. The next day, sections were incubated with ABC-HRP kit (Vectastain) for 30 min, followed by 5 min incubation with DAKO-DAB substrate (EnVision). Slides were dehydrated using standard protocols and counterstained with haematoxylin.

Paraffin-embedded blocks were cut into 4-μm thick sections. E1A and HA staining were performed as previously published,¹⁴ (link)^,21 (link) using the anti-Ad2/5 E1A antibody (1/200; Santa Cruz Biotechnology, SC-25) and B-HABP (5 μg/mL; Amsbio, AMS.HKD-BC41). For human CD3 detection, FLEX Polyclonal Rabbit Anti-Human CD3 (IR503, Agilent DAKO) was diluted 1:10 with DAKO antibody diluent (Dako – Agilent, S0809) for 120 min at room temperature. The secondary antibody used was a BrightVision poly-horseradish peroxidase (HRP)-anti-rabbit immunoglobulin G (IgG) that was biotin-free, ready to use (Immunologic, DPVR-110HRP) incubated 45 min. Antigen-antibody complexes were reveled with 3-3′-diaminobenzidine (K3468, Dako). H&E staining was performed according to standard procedures. Masson trichromic staining was performed using the Accustain Trichrome Stain Kit (Sigma Aldrich) according to the manufacturer’s indications. The percentage of stained areas in IHC images were quantified after color deconvolution by ImageJ FIJI⁴⁴ (link) software.