Fitc conjugated donkey anti rabbit igg

Primary lung fibroblasts were cultured in a dish at 37 °C, and the mitochondria were stained with 100 nM MitoTracker Red (Invitrogen) for 20 min. Then, the cells were fixed with 4% (v/v) paraformaldehyde (PFA) for 45 min, permeabilized in ice-cold buffer (0.1% [w/v] sodium citrate and 0.1% [v/v] Triton X-100 in distilled water) for 3 min, blocked with Dulbecco’s phosphate-buffered saline with 1% (w/v) BSA for 1 h; and incubated with an anti-OAT antibody (1:200; Santa Cruz, #SC-374243) overnight at 4 °C. After washing, the cells were incubated with a fluorescein isothiocyanate (FITC)-conjugated anti‐mouse antibody (1:1000; Abcam, #ab6785) for 1 h at room temperature and mounted in fluorescence medium containing 4ʹ,6-diamidino-2-phenylindole (DAPI; Abcam). To measure the expression of TGF-β1 in BLM-treated OAT-knockdown cells, the cells were subjected to double immunofluorescence staining for TGF-β1 (1:100; Thermo Fisher Scientific, #MA5-15065) and OAT (1:100). FITC-conjugated donkey anti-rabbit IgG (1:1000; Abcam, #ab150061) and PE-conjugated goat anti-mouse IgG (1:1000; Abcam, #ab97024) served as the secondary antibodies. All the stained images were captured using a Leica DMi8 confocal laser microscope (Leica Microsystems; Wetzlar, Germany).

First, cells in slides were washed with PBS, fixed with 4% paraformaldehyde for 30 min, then refrigerated in PBS until further use. Immediately prior to staining, the cells were washed with PBS, permeabilized with PBS containing 0.05% Triton X-100 and 10% goat serum for 5 min, and then incubated with anti-ADRP antibody (1:200 dilution; Abcam, Cambridge, MA, USA) overnight at 4°C. Next day, the cells were washed with PBS, incubated with a fluorescein isothiocyanate (FITC)-conjugated donkey anti-rabbit IgG (1:1000 dilution; Abcam, Cambridge, MA, USA) for 1 h at 37°C, followed by washing with PBS for three times, and the nuclei were then counterstained with 4, 6-diamidino-2-phenylindole (DAPI, Sigma, St. Louis, MO, USA). Bound antibodies were viewed under a confocal microscope (Olympus Fluoview 1000, Japan).

To confirm that injected MSCs^ATP7B had differentiated into normal hepatocytes, GFP/CK-18 double fluorescence intensity was assessed using a previously described protocol [31] (link). Briefly, liver tissues were embedded in tissue freezing medium (Tissue-Tek OCT Compound, Sakura Finetek, Torrance, CA, USA) and frozen at −80°C. Frozen sections were cut at 4 µm, fixed in cold acetone and then blocked in bovine serum albumin (BSA). Anti-CK-18 rabbit monoclonal antibody (Abcam, Cambridge, MA, USA) and anti-GFP mouse monoclonal antibody (Abmart, Shanghai, China) diluted, respectively, to 1∶100 and 1∶500 in PBS containing 1% BSA were applied to the blocked sections for 16 hours at 4°C. Negative controls were incubated with PBS containing 1% BSA instead of primary antibody. After washing in PBS, FITC-conjugated donkey anti-rabbit IgG (1∶1000, Abcam, Cambridge, MA, USA) and PE-conjugated goat anti-mouse IgG (1∶400, Santa Cruz, CA, USA) were applied for 1 hour at room temperature.