T7471

Primary BEC were cultured on glass chamber slides (80827, ibidi GmbH, Germany). Cells were starved in media without serum and exposed to stimulants for 30 min. Then, cells were fixed in 4% paraformaldehyde in PBS pH 7.4 for 10 min at room temperature and washed 3× with ice-cold PBS for 5 min. Cells were permeabilized with 0.1% Triton X-100 in PBS for 10 min and stained with Texas Red-X Phalloidin for 20 min at room temperature (T7471, ThermoFischer Scientific, Waltham, MA) or alternatively incubated overnight at 4 °C with primary antibody against PAR1 (sc-13503, Santa Cruz Biotechnology Inc., Dallas, TX) and PAR4 (sc-1666, Santa Cruz Biotechnology Inc., Dallas, TX). The samples were subsequently washed and incubated with secondary antibody (A21203, Life Technologies, Eugene OR) for 1 h at room temperature, and 5 min with DAPI (Sigma Aldrich, St. Louis, MO). Cells were imaged at the Stanford University Cell Sciences Imaging Facility on a Zeiss 880 confocal microscope using a ×40 objective.

Hepatic paraffin sections (3 μm) of CCl₄-induced liver fibrosis rat models were prepared for the IF staining for progerin, vWF, and LC3B. The primary antibodies included anti-progerin (1:50), anti-vWF (1:200), and anti-LC3B (1:200). Subsequently, the sections were exposed to the secondary antibodies and DAPI. The sections were visualized with the fluorescence microscope.
The treated LSECs in confocal dishes were fixed with 4% paraformaldehyde and then permeabilized with 0.1% Triton X-100 and blocked with 3% BSA. Next, LSECs were incubated with primary antibodies, including anti-acetyl Lysine (1:200), anti-progerin (1:50), and anti-LC3B (1:200), followed by the secondary antibodies and DAPI. Lastly, LSECs were stained with a phallotoxin (Thermo, #A22287 and #T7471) to detect F-actin. The positive cells were observed by the confocal microscopy and quantified by Image J V1.8.0 software. The experiment was repeated three times.

Primaries: chicken anti-GFP (1:1000, Abcam ab13970, RRID:AB_300798); rabbit anti-pS19-MRLC (1:50, CST 3671); mouse anti-Delta extracellular domain (1:100, DSHB c594.9b, RRID:AB_528194); mouse anti-Notch extracellular domain (1:200, DSHB c458.2H, RRID:AB_528408); guinea pig anti-Delta ECD (1:2000, M. Muskavitch). Secondaries: Rhodamine Red-X anti-guinea pig (1:2000, Jackson ImmunoResearch Labs #106-295-003, RRID:AB_2337428) and Cy5 anti-mouse (1:2000, Jackson ImmunoResearch Labs #115-175-146, RRID:AB_2338713); AlexaFluor 488 anti-chicken (1:1000, ThermoFisher Scientific A-11039, RRID:AB_2534096) and AlexaFluor 568 anti-rabbit (1:1000, ThermoFisher Scientific A-11011, RRID:AB_143157); Texas Red-X Phalloidin (1:500, ThermoFisher Scientific T-7471) were used to visualize F-actin. Nota were mounted in 50% glycerol or Vectashield (with DAPI, Vector Laboratories) for labeling nuclei.