Alexa 488 conjugated goat anti rabbit igg secondary antibody

Select animals of Pah^+/+ and Pah^{Δexon1/Δexon1} genotype, as well as gene therapy treated Pah^{Δexon1/Δexon1} animals, underwent saline-paraformaldehyde perfusion in preparation for liver fixation and immunohistochemistry (Fig. 3C and Fig. 4). Under deep isoflurane inhaled anesthesia, mice were perfused with 0.9% saline via cardiac infusion to clear the hepatic circulation of blood. Excised biopsies from each liver lobule were then fixed for 24 h in fixation solution (4% paraformaldehyde in phosphate buffered saline) followed by immersion in 25% buffered sucrose for an additional 24 h. Tissues were embedded in optimal cutting temperature media, sliced into 15 μm sections, and mounted on glass slides. Sections were treated (following blocking) with rabbit polyclonal anti-PAH antibody (1:200 dilution, BS3704 PAH (R400) from Bioworld, Inc.) and incubated for 2 h at room temperature. After appropriate washes, sections were treated with Alexa 488-conjugated goat anti-rabbit IgG secondary antibody (1:150 dilution, A11008 Alexa Fluor 488 goat anti-rabbit IgG (H + L) from Invitrogen) and incubated for one hour at room temperature. After washing, cover slips were mounted with Vectashield mounting medium containing DAPI. Images were captured on a Nikon H550L fluorescence microscope.

Select animals from each experimental cohort underwent saline-paraformaldehyde perfusion in preparation for brain fixation and immunohistology. Under deep isoflurane inhaled anesthesia, mice were perfused with 0.9% saline via cardiac infusion to clear the cerebral circulation of blood and then were perfused with an equal volume of fixative solution (4% paraformaldehyde in phosphate buffered saline). Excised whole brains were then fixed for 24 hours in the same fixation solution followed by dehydration in 25% buffered sucrose for an additional 24 hours. 15 micrometer coronal frozen sections were then cut from the regions of the striatum using a cryostat and mounted on glass slides. Sections were treated (following blocking) with rabbit polyclonal anti-TH antibody (1:200 dilution, EMD Millipore Corp. AB152) and incubated for 2 hours at room temperature. After appropriate washes, sections were treated with Alexa 488-conjugated goat anti-rabbit IgG secondary antibody (1:150 dilution, Thermo Scientific, A27034) and incubated for one hour at room temperature. After washing, cover slips were mounted with Vectashield mounting medium containing DAPI. Images were captured on a Nikon H550L fluorescence microscope.