(A–F) Localization of transgene protein expression was determined by immunofluorescent microscopy. Isolated platelets from FVIII^null (A–C) and 2bF8^trans mice (D–F) were immunostained for either human FVIII (hFVIII; A and D) or mouse VWF (B and E). The 2 images were merged in C and F, showing that in the platelets of 2bF8^trans mice (F) VWF and FVIII were colocalized (yellow). (G–K) Colocalization of human FVIII and mouse VWF was confirmed by electron microscopy. Isolated platelets from FVIII^null mice (G and H) and 2bF8^trans mice (I–K) were immunostained for human and mouse VWF. FVIII was probed with 5 nm colloidal gold and VWF with 10 nm colloidal gold; representative gold particles are indicated by arrows and arrowheads, respectively. The results show FVIII was stored together with VWF in platelet α-granules of 2bF8^trans mice (I–K). (L–Q) Confocal microscopy detected no human FVIII in mononuclear cells from transgenic mice. Isolated platelets and mononuclear cells from 2bF8^trans mice were immunostained for human FVIII. Nonspecific isotype-matched primary Ab was used for background staining. (R) Quantitative evaluation of FVIII:C levels in mouse mononuclear cell lysates. Platelets and mononuclear cells were isolated and lysed in 0.5% CHAPS. FVIII:C in lysates was determined by chromogenic assay. No FVIII:C was detected in mononuclear cell lysates from either 2bF8^trans or control mice (i.e., WT and FVIII^null mice). FVIII:C was detected in platelet lysates from 2bF8^trans mice. Scale bars: 8 μm (A–F and L–Q); 0.2 μm (G–K).