Goat anti mouse igg alexa fluor568

Liver sections (6 μm) were deparaffinized and rehydrated with toluene and ethanol by routine procedures. Antigen retrieval was performed by boiling slides in 10 mM citrate buffer, pH 6.0, 0.02% Tween-20 (Arg1 staining) or Tris/EDTA buffer, pH 9.0, 0.05% Tween-20 (CD4 staining). After PBS rinsing, sections were permeabilized with 1 × PBS + 0.2% Triton X-100 for 10 min at room temperature then blocked with 2% normal goat serum (Cedarlane) in PBS for 30–45 min. Sections were incubated with rabbit polyclonal anti-Arg1 (1:100; Abcam #ab91279), rabbit monoclonal (EPR19514) anti-CD4 (1:250; Abcam #ab183685) in 1–2% goat serum, and mouse monoclonal anti-glutamine synthetase (1:200; Abcam #ab64613) in serum, for 1 h at room temperature or overnight at 4 °C. Secondary antibodies, goat anti-rabbit IgG FITC (Arg1; Cedarlane), goat anti-rabbit IgG (H + L) Alexa Fluor 488 (CD4; 1:1000; Life Technologies #A11008) and goat anti-mouse IgG AlexaFluor 568 (GS; Life Technologies) were used. Slides were then dehydrated, mounted with ProLong Gold antifade reagent with DAPI (Invitrogen), and stored at room temperature until analysis. Visualization was performed with a fluorescent microscope (Leica, DM IRB, Richmond Hill, ON). Separate H&E sections, from both the gene therapy and hepatocyte transplantation studies, were examined by a pathologist blinded as to the identity of the samples.