Figure 3: Effects of fixative techniques on immunofluorescent staining for hF.IX and CD8 with a fixed exposure time. Skeletal muscle from various C57BL/6 mice expressing hF.IXwere optimally cryopreserved in LqN2-cooled isopentane, serial sectioned (10 μm) and subsequently fixed as indicated.(A) Unfixed tissue, (B) fixed with acetone for 3 minutes at room temperature, (C) fixed with acetone for 3 minutes at -20° C, or (D) fixed with 4% PFA for 10 minutes followed by methanol for 10 minutes at room temperature. Following fixation, sections were stained for hF.IX (red) and CD8 (green), and mounted with a media containing DAPI (blue). Exposure times for all slides were optimized using the unfixed slide. Asterisk (*) identifies the same cell in each serial section.