Hydrogen peroxide

RNA-ISH was performed on cytocentrifuged single cells using the RNAscope Multiplex Fluorescent Assay v2, and on paraffin-embedded 5 μm tissue sections using the RNAscope 2.5 HD Reagent Kit and RNAscope 2.5 HD Duplex Reagent Kit according to the manufacturer’s instructions (Advanced Cell Diagnostics). Cytospin slides were rehydrated with graded ethanol (100% 1 min, 70% 1 min, 50% 1 min), permeabilized with PBS + 0.1% Tween 20 (PBST) at RT for 10 min, incubated with hydrogen peroxide (Advanced Cell Diagnostics) at RT for 10 min, followed by incubation with 1:15 diluted protease III at RT for 10 min. Tissue sections were deparaffinized with xylene (2 changes × 5 min) and 100% ethanol (2 changes × 1 min), and then incubated with hydrogen peroxide for 10 min, followed by target retrieval in boiling water for 15 min, and incubation with Protease Plus (Advanced Cell Diagnostics) for 15 min at 40°C. Slides were hybridized with custom probes at 40°C for 2 h, and signals were amplified according to the manufacturer’s instructions. The stained sections were scanned and digitized using an Olympus VS120 light or fluorescent microscope with a 40X 1.35 NA objective and Olympus confocal microscope with a 40X 0.6 NA or 60X 1.4 NA objective.

At 30 or 60 days after the procedure, the dogs were euthanized, and livers were harvested and sectioned according to an alphanumerical grid. Selected 7-μm cryosections were dehydrated in 50%, 70%, or 100% ethanol and stored in 100% ethanol overnight. Hydrogen peroxide (Advanced Cell Diagnostics [ACD]) was added for 10 minutes, and then protease inhibitor IV (ACD) was added for 30 minutes at room temperature. The slides were then subjected to the RNAscope 2.5 HD – Red Assay according to standard protocols from ACD. Additional details are provided in supplemental Methods.