Immunohistochemical staining (IHC) of factor IX. Hemostatically normal C57BL/6 mice received IA injection of 2.5 × 109 vg AAV2, AAV5, or AAV8 expressing hFIX into the left knee joint capsule. Four weeks later, the AAV-treated knee and untreated control knee were collected, and immunostaining performed for hFIX using polyclonal anti-FIX (A) or using monoclonal antifactor IX A-7, specific for γ-carboxylated factor IX (B). HRP stain appears brown in FIX-expressing cells. Hematoxylin was added as counterstain in all images. (A) Control knee has primary (antifactor IX) antibody omitted and subsequent staining steps performed, demonstrating no background HRP (brown stain). Panels labeled AAV2, 5, and 8 cartilage demonstrate HRP staining cells within ssAAV2, 5, and 8-CBA-hFIX-transduced cartilage. Panels labeled AAV2, AAV5, or AAV8 synovium demonstrate HRP staining cells within ssAAV2, 5, and 8-CBA-hFIX-transduced synovium. (B) Immunostaining performed using monoclonal anti-FIX A-7 antibody. Control (negative) has primary A-7 antibody omitted and subsequent staining steps performed. Representative images of ssAAV8-CBA-hFIX transduced cartilage and ssAAV8-CBA-hFIX transduced synovium are shown after A7-HRP labeling. Pink arrows represent individual factor IX-staining chondrocytes within cartilage; black arrows, transduced FLS within the synovial lining (original magnification ×400). N = 3 to 5 animals per treatment group. (C) Quantitative analysis of cells in cartilage and synovium expressing hFIX after AAV2-, AAV5-, or AAV8-CBA-hFIX transduction. Data are represented as percentage of hFIX positive staining cells; error bars represent SD (n ≥ 4 animals/group).
