Hpa005692

Samples were denaturated by incubating at 95 °C for 5 min in a non-reduced sample buffer and separated by using SDS-PAGE (pre-cast 8% × 16% gradient gels, Biorad, Hercules, CA, USA). The gels were then stained with 0.1% Commassie blue. To determine the presence of FIX and Tf domain in the fusion protein, anti-FIX and anti-Tf Western blot was performed. Antibody against human FIX (ab124815, Abcam, Cambridge, MA, USA) and antibody against human Tf (HPA005692, Sigma-Aldrich, St. Louis, MO, USA) were used as the primary antibodies, while horseradish peroxidase-conjugated anti-rabbit IgG antibody (#7074, Cell Signaling, Danvers, MA, USA) was used as the secondary antibody. The peroxidase activity was detected by maximun sensitivity chemiluminescence (#34095, Thermo Scientific, Waltham, MA, USA) for visualization.

For immunohistochemistry, 3–4-μm-thick liver sections were deparaffinized and rehydrated, and a citrate-based antigen retrieval was performed (Vector H-3300, Vector Laboratories, Petersborough, UK) for 30 min, as recommended by the supplier. The immunodetection was carried out with anti-transferrin as a primary (11,000 dilution; Sigma; HPA005692, Stockholm, Sweden) and a biotin-conjugated anti-rabbit secondary antibody (Vector BA1000). The signal was visualized with peroxidase-labeled streptavidin and the 3,3′-diaminobenzidine as a substrate (Vector SK4100).