Figure 4: Effects of fixative techniques on immunofluorescent staining for hF.IX and CD8 with variable exposure times. Serial sections (10 μm) of skeletal muscle from various C57BL/6 mice expressing hF.IX were optimally cryopreserved in LqN2-cooled isopentane were fixed as indicated. (A,E) Unfixed (B,F) PFA/MeOH fixed, (C,G) acetone at room temperature fixed, or (D,H) acetone at -20° C. Tissues were stained for hF.IX (red), CD8 (green), and DAPI (blue). The software automatically optimized exposure time for each slide, and the times used are displayed below each image in seconds. (A-D) Images were taken for all 3 channels using a 10x objective. (E-H) Images were taken only for the green channel (CD8) using a 20x objective.