Histology and Immunostaining of liver tissues
Histology staining was performed as described previously with few modifications (Qiu et al., 2021). Briefly, Liver tissues from mouse were collected from all the treated groups after postmortem and fixed in ice cold 4% paraformaldehyde (PFA) overnight at 4°C. The following day samples the sections were paraffin embedded and were cut in 4um thickness following deparaffinization, the sections were stained with hematoxylin for (5-15 mins) washed and counter stained with eosin. The slides were quickly dehydrated and sealed using mineral oil. The images were documented at 20X magnification on Olympus BX53.
For immunostaining 4-um liver sections were cut and mounted on slides from all the groups the sections were deparaffinized with xylene and dehydrated in ethanol. The procedure was performed according to manufacture instructions (ab209101, Rabbit specific IHC polymer detection kit). The sections were immersed in 0.3% H2O2/methanol was used for 30 mins to reduce nonspecific staining to remove exogenous peroxidase avtivity, next the sections were incubated in primary antibody Phospho-AMPKa (Thr 172) (Rabbit mAb# 2535, cell signaling) overnight at 4°C in 3% BSA in PBS, followed by PBS washing, the slides were stained with anti-rabbit IgG. DAB was used for color rendering, and counter stained with hepatotoxicant, dehydrated, and sealed. Images were documented using Olympus BX53 at 20X magnification.