The test and negative control slides used here were prepared as described in Daenzer et al [7 (link)]. Specifically, test slides included 5 micron sections of fixed liver tissue derived from GALT-null rats that had been administered an scAAV9-HA.hGALT vector either 14 or 30 days prior to euthanasia. Negative control slides included 5-micron sections of fixed liver tissue from GALT-null rats that had been administered vehicle alone (phosphate-buffered saline, PBS). The individual rats whose samples are presented here include: Rat A (FKRC311.6), Rat B (FKRC297.2), Rat C (FKRC297.12), Rat D (FKRC297.10) and Rat E (FKRC297.9).
Immunohistochemistry (IHC) was performed using an anti-HA monoclonal antibody (Cell Signaling Technology #3724) as primary at 1:1000 dilution, and a peroxidase-coupled goat anti-rabbit IgG (H+L) (Vector Labs PI-1000-1) as secondary. Antibody binding was visualized as brown color by reaction of the coupled peroxidase with 3,3′-diaminobenzidine (DAB; Vector Labs, SK-4100), as recommended by the manufacturer. All slides were also treated with hematoxylin which nonspecifically stained all cells a light blue color. Mounted slides were scanned on a Hamamatsu Nanozoomer 2.0 HT whole slide scanner at 40X and viewed using the free Hamamatsu software (NDP viewer).
Immunohistochemistry (IHC) was performed using an anti-HA monoclonal antibody (Cell Signaling Technology #3724) as primary at 1:1000 dilution, and a peroxidase-coupled goat anti-rabbit IgG (H+L) (Vector Labs PI-1000-1) as secondary. Antibody binding was visualized as brown color by reaction of the coupled peroxidase with 3,3′-diaminobenzidine (DAB; Vector Labs, SK-4100), as recommended by the manufacturer. All slides were also treated with hematoxylin which nonspecifically stained all cells a light blue color. Mounted slides were scanned on a Hamamatsu Nanozoomer 2.0 HT whole slide scanner at 40X and viewed using the free Hamamatsu software (NDP viewer).