Polyfreeze

Livers from infected mice after 21 d.p.i. or non-infected mice (control) were harvested, embedded in Polyfreeze (Sigma-Aldrich, United States), and snap-frozen in nitrogen. Sections were cut at a thickness of 10 μm, stained with hematoxylin and eosin and analyzed under light microscope Zeiss Axio LabA1.

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).

Post fixation in 10% formalin, the tissues were embedded in Optimal Cutting Temperature (OCT) compound (PolyFreeze, Sigma-Aldrich) in plastic Cryomolds^® (VWR, PA, USA) using chilled isopentane. Transverse sections (15 µm) were obtained by sectioning with a Leica CM1900 cryostat. The sections were transferred to SuperFrost Plus Adhesion slides (Fisher Scientific) and stored at −20°C.
Prior to staining, the tissue sections were encircled with a PAP pen (ImmEdge, Vector laboratories, CA, USA). PBS was used to hydrate the sections before a 15-min permeabilization step with 0.1% Triton X-100 in PBS (PBS-T). Tissue sections were then blocked for 30-40 min at RT in blocking buffer (0.3% Triton X-100 and 3% donkey serum in PBS) followed by incubation with primary antibodies (see Table S1 for details) in blocking buffer overnight at 4°C. After this incubation, the sections were washed three times with PBS-T (5 min per wash) and incubated with fluorophore-tagged secondary antibodies and phalloidin (Table S1) in blocking buffer for 40-60 min at RT. Sections were then washed three times with PBS-T (5 min per wash) and mounted using VECTASHIELD Plus Mounting Medium with DAPI (Palex). After placing coverslips, the corners of the slides were sealed with a transparent enamel sealant.