For immunostaining of liver sections for FIX expression, murine liver was embedded in OCT media (Polyfreeze, Sigma-Aldrich) and after sectioning was fixed with 4% paraformaldehyde. Tissue sections were blocked with 10% normal donkey serum (Santa Cruz Biotechnology, Dallas, TX, USA) and then incubated with goat antihuman FIX (Affinity Biologicals, Hamilton, ON, Canada) overnight at 4 °C. Samples were probed with the donkey antigoat Cy3 antibody (Jackson ImmunoResearch, West Grove, PA, USA). For nuclear staining, 4,6-diamidino-2-phenylindole was used. Images were acquired in a Leica DMi8 confocal microscope (Wetzlar, Germany).
For immunostaining of retinal sections, eye balls from the control and AAV2-treated mice were harvested after 6 weeks of gene transfer.27 (link) Cryosectioned retinal sections were permeabilized with 0.5% Triton X-100 for 15 min, followed by incubation with the blocking agent (10% normal goat serum, Abcam, Cambridge, UK) for 1 h. The retinal sections were then incubated with a mouse monoclonal antibody to GFP at a 1:50 dilution (Abcam) in 10% normal goat serum and further stained by the FITC-labeled rabbit antimouse antibody (Abcam, Cambridge, UK). For nuclear staining, we used 4,6-diamidino-2-phenylindole. The retinal sections were imaged by confocal microscopy (Leica DMi8).
For immunostaining of retinal sections, eye balls from the control and AAV2-treated mice were harvested after 6 weeks of gene transfer.27 (link) Cryosectioned retinal sections were permeabilized with 0.5% Triton X-100 for 15 min, followed by incubation with the blocking agent (10% normal goat serum, Abcam, Cambridge, UK) for 1 h. The retinal sections were then incubated with a mouse monoclonal antibody to GFP at a 1:50 dilution (Abcam) in 10% normal goat serum and further stained by the FITC-labeled rabbit antimouse antibody (Abcam, Cambridge, UK). For nuclear staining, we used 4,6-diamidino-2-phenylindole. The retinal sections were imaged by confocal microscopy (Leica DMi8).