FISH analysis of cFVIII RNA in dog liver. RNA in situ hybridization was performed on liver sections biopsied from Junior 8 months after injection of AAV-cFVIII-SQ at a dose of 2.7 × 10¹³ vg/kg. The RNA signal (green staining) was detected using a cFVIII antisense probe cocktail (A). Counter-staining (blue staining) of the same section with 4′, 6-diamidino-2-phenlindole is shown (B). No signal was detected when the sections were pretreated with RNase (data not shown), when a cFVIII sense probe was used (C), or when a naive Hem A liver was analyzed (D). Cells were analyzed under a × 40 objective by confocal microscopy.