Hematoxylin and eosin h e stain

Following fixation, cells were washed and assessed by standard Hematoxylin and Eosin (H&E) Stain (according to manufacturer’s protocol; Sigma, USA). Periodic Acid-Schiff (PAS) staining was used to assess for glycogen accumulation within the cells using the PAS kit (395B-1KT, Sigma-Aldrich) without pre-digestion with diastase. Alkaline Phosphatase (AP) staining was used to assess for differentiation of sinusoidal trophoblast giant cells. Briefly, cells were rinsed in PBS, incubated in wash buffer (pH 9.5) and alkaline phosphatase activity was detected by using NBT/BCIP substrate to form a blue precipitate. The reaction was stopped in PBS and cells were counterstained in Nuclear Fast Red (Vector Labs, USA) for 1 min, followed by dehydration through a graded ethanol series. Images were collected using a Leica DMR light microscope (fluorescence and bright-field) and an EVOS XL Core microscope (bright-field; Invitrogen, USA).

2.2-Diphenyl-1-picryl hydrazyl (DPPH), butylated hydroxytoluene (BHT), d(+)-catechin, gallic acid, tannic acid, sodium carbonate, nitrobluetetrazolium (NBT), xanthine, xanthine oxidase, and Folin-Ciocalteu reagent, iron(III) chloride (FeCl₃), hydrogen peroxide (H₂O₂), ascorbic acid were all purchased from Sigma-Aldrich (St. Louis, MO, USA) or Merck & Co (Darmstadt, Germany). For in vitro studies, bovine serum albumin, Dulbecco’s Modified Eagle Medium (DMEM), fetal bovine serum (FBS), Dulbecco’s phosphate buffered saline (DPBS), penicillin, and streptomycin were purchased from Hyclone (Logan, UT, USA). Oligo primers were purchased from Macrogen (Seoul, Korea). For in vivo studies, silymarin, olive oil, CCl₄, Oil Red O, hematoxylin and eosin (H&E) stain, triethanolamide, 5,5’-dithiobis(2-nitrobenzoic acid) (DTNB), glutathione (GSH), superoxide dismutase (SOD), 2-thiobarbituric acid (TBA), and sodium azide were purchased from Sigma-Aldrich.