Picosirius red stain kit

The livers were fixed overnight in 4% paraformaldehyde, processed to 70% EtOH in saline, embedded in paraffin, and sectioned at 7 μm. Slides were stained with hematoxylin-eosin (ab245880, Abcam) for histologic examination. For ki67 (ab16667, Abcam) staining, slides were deparaffinized and rehydrated through an ethanol gradient to 1× phosphate-buffered saline. Antigen retrieval was performed at high pressure and high temperature in sodium citrate pH 8 buffer. The samples were blocked for 1 hour at room temperature. The samples were then incubated overnight at 4°C with primary antibody diluted in blocking buffer. The next day, the samples were washed and incubated with secondary antibody diluted in blocking buffer for 4 hours at room temperature, counterstained with Mayer’s hematoxylin, dehydrated through an ethanol series, and mounted with Cytoseal 60 (Thermo Scientific, 8310-4). The percentage of ki67-positive hepatocytes was calculated by manually counting five ×20 image fields from an animal (n = 3 for each arm).^{18 ,20} (link) Sirius Red staining was performed using the Picosirius Red Stain Kit from Polysciences (24901) following manufacturer’s instructions. Images were taken using a Nikon Eclipse 90i Microscope and a Nikon Eclipse Ti Microscope.