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.