Ab3648

Tissues were formalin-fixed, paraffin-embedded (FFPE), cut, and stained with H&E, as previously described.⁶³ (link) For immunohistochemical staining, 4- to 5-μm sections of the FFPE hearts were cut and deparaffinized in xylene and rehydrated in graded alcohols.⁶⁴ (link) Endogenous peroxidases were blocked by incubating sections in methanol with 3% hydrogen peroxide for 20 min. Antigen heat retrieval was performed in citric buffer for 12 min, followed by a cooling period of 15 min. Slides were then incubated for 30 min with Roti-Immunoblock (Roth) and goat serum to block non-specific antibody binding. Sections were incubated overnight at 4°C with monoclonal mouse anti-adenovirus antibody (ab3648, Abcam). The antibody recognizes the hAd5 E1A protein.⁶⁵ (link) Slides were then incubated with a biotinylated secondary goat anti-mouse antibody (dilution 1:200 each; Vector Laboratories) for 30 min at room temperature. Immunolabeling was performed by an avidin-biotin-immunoperoxidase system (Vectastain Elite ABC Kit; Vector Laboratories). Diaminobenzidine tetrahydrochloride (DAB; Merck) was used for antigen visualization of viral protein.