Figure 4.

hFIX expression and CD8⁺ T cell infiltration in transduced muscles. Skeletal muscles from hemophilia B (C3H/HeJ F9^−/Y) mice were harvested, cryosectioned, and stained for hFIX (red) and CD8 (green) 4 weeks after AAV1-CpG⁻ or AAV1-CpG⁺ injection, as described in Figure 3A. Muscles section was entirely scan and images were taken for both channels using a 40× objective with ZEISS Microscopy. (A) Sequential scan image of transduced muscle from AAV1-CpG⁺ (left) or AAV1-CpG⁻ (right) injected mice shown for individual channel or merge image. (B) Numbers of infiltrating CD8⁺ T cells in transduced muscle as quantified by Fiji-ImageJ software after generating a grid (area per point, 1200 square pixels). Whole muscle sections were quantified. (C) Fluorescent signal for hFIX quantified as mean gray value with Fiji-ImageJ software. Representative images from five mice are shown for each condition. The scale bar represents 100 μm. Data are average ± SEM of at least five animals per group. Each circle represents an individual animal. Statistically significant differences are indicated. *P < 0.05.