Immunostaining of hFVIII. Localization of transgene protein expression was determined by immunofluorescent confocal microscopy. Endothelial cells were isolated from lung (A) and heart (B) of neonatal mice by immunoselection using Dynabeads M-450 sheep anti-rat IgG beads coated with purified rat anti-mouse CD31 (PECAM-1) antibody. Cells were cultured in vitro for 4 days and then immunostained for either hFVIII using Conan-Alexa 488 or mouse VWF using Dako-Alexa 568. Transgene protein hFVIII was detected in endothelial cells from both lung (Ai-vi) and heart (Bi-vi) of T2F8^Tg mice and was colocalized with mouse VWF in Weibel-Parade bodies as shown in yellow in merge images (iii, vi, xi, and xv). No hFVIII was detected in the endothelial cells from FVIII^null mice (Avii-ix,Bvii-x) These results demonstrate that targeting FVIII expression to endothelial cells results in FVIII storage together with VWF in endothelial cell WPB.