Typhoon fla 9410 laser scanner

Western blot assays were conducted on mouse plasma samples collected from control and mutant mice treated with the hFIX vectors using a monoclonal anti-human Factor IX antibody (Haematologic Technologies, Essex Junction, VT, USA, Cat. No. AHIX-5041) and an Alexa Fluor 568 fluorescent dye conjugate (Life Technologies, Carlsbad, CA, USA, goat anti-mouse, Cat. No. A-21124). Citrated plasma samples were subjected to electrophoresis through 12% polyacrylamide gels, and transferred to polyvinylidene fluoride membranes (EMD Millipore, Billerica, MA, USA; Cat. No. IPVH00010). After blocking with 5% nonfat dried milk the blots were incubated overnight with primary antibody diluted 1:2000, and then fluorescent secondary antibody diluted 1:2000. The blots were washed and then imaged using a Typhoon FLA 9410 laser scanner (GE Healthcare Life Sciences, Pittsburgh, PA, USA). The hFIX immunoblots were stripped and reprobed with the goat anti-mouse serum albumin antibody (Bethyl Laboratories Inc., Montgomery, TX, USA, Cat. No. A90-2394) at a 1:1000 dilution. The membranes were washed and incubated with a donkey anti-goat 568 Alexa Fluor 568-conjugated secondary antibody diluted 1:2000. Densitometry analyses of western blot bands were evaluated using ImageJ software (NIH, Bethesda, MD, USA).