Vectamount aq aqueous mounting medium

Fetal liver tissue samples from the left lobe were fixed in 10% formalin for 24 h followed by storage in 70% EtOH. Histology was performed by the OUHSC Stephenson Cancer Tissue Pathology Core. In brief, samples were paraffin-embedded and sectioned for H&E and picrosirius red staining. Fresh-frozen liver from the left lobe was fixed in OCT compound, sectioned, and fixed with formalin for 5 min and washed with PBS. LipidSpot lipid droplet stain (Biotium, Fremont, CA) was added to the sections and incubated for 20 min. Sections were washed with PBS, counterstained with DAPI, and mounted using VectaMount AQ aqueous mounting medium (Vector Labs, Burlingame, CA). Slides were visualized using a Cytation 5 microscope and Gen5 imaging software (Agilent, Santa Clara, CA).

Routine H&E staining of liver frozen sections was performed as described previously (32 (link)). Briefly, the sections were fixed with formalin overnight at room temperature and stained with Harris modified hematoxylin/Eosin-Phloxine solution (Newcomer Supply, #1201 and #1082A). The lipid content of liver specimens was analyzed by staining with Oil Red O, and the nuclei were counterstained with hematoxylin. Slides were mounted with VectaMount™ AQ aqueous mounting medium (Vector Laboratories, H-5501) and imaged using upright Olympus microscope with 4x and 20x objectives.

Hepatocytes were cultured in glass bottom dishes (Cellvis-D35-14-0-N, Mountain View, CA, USA) and treated with senescence inducers for 24 h. Cells were fixed with 4% paraformaldehyde for 15 min followed by permeabilization with 0.2% Triton X-100 for 15 min at room temperature. Antigens were blocked with 2% normal goat serum and 1% bovine serum albumin (BSA) for 1 h in a humidified chamber at room temperature to prevent non-specific binding of the antibodies. Then, cells were incubated with primary antibodies (table S3) diluted in Antibody Diluent (Thermo Fisher Scientific-003218, Waltham, MA, USA) overnight (16 h) in a humidified chamber at 4 °C. Next day, the primary antibodies were washed off and the samples were incubated with fluorescently labeled secondary antibodies (table S4). Then the DNA was stained with DAPI (Sigma-Aldrich-D9542, St. Louis, MO, United States) and the F-actin was stained with Phalloidin-iFluor 488 (Abcam- ab176753, Cambridge, United Kingdom). Samples were mounted with VectaMount^® AQ Aqueous Mounting Medium (Vector laboratories- H-5501-60, Newark, CA, USA) and imaged with Axio Observer 7 (Carl Zeiss Microscope). Fluorescence intensity or the number of nuclei was measured with Image J software.