Nucblue fixed cell readyprobes reagent dapi

iHEPs were treated +/−20 nM staurosporine (Generon) for 2 h, after which the cells were fixed in 4% PFA (Sigma-Aldrich) in PBS for 15 min. The fixed cells were then blocked for 1 h in blocking buffer (0.2% Triton X-100 (Sigma-Aldrich), 3% Donkey-Serum (Sigma-Aldrich) and 1% BSA (Sigma-Aldrich) in PBS) at room temperature. Next, the cells were stained with a 1:25 dilution of human MRP2 primary antibody (Abcam; ab3373) in blocking buffer overnight at 4 °C. The following day, the cells were stained with a 1:500 dilution of AlexaFluor 555 donkey anti-mouse antibody (Invitrogen; A-31570) in PBS for 2 h at room temperature, followed by a 5-min incubation with NucBlue Fixed Cell ReadyProbes Reagent (DAPI; Thermo Fisher Scientific) at room temperature. Cell nuclei (blue) and MRP2 expression at the canalicular membrane (red) were captured in images taken on an LSM 880 confocal microscope (ZEISS). Images were analysed using Fiji^{45 (link)} as above to determine integrated density of MRP2 staining at the canalicular membrane, total canalicular area, average canalicular area and canalicular count. The statistical significance of experimental data was assessed by Student’s test.

Cells were seeded in 8-well imaging chambers at a density of 30K cells/chamber and grown for 48 h with daily feeding. After 48 h, cells were washed once in PBS and fixed in 4% paraformaldehyde (20 min, RT). Fixed cells were washed in PBS three times and incubated in primary antibody ICC antibody diluent [0.05% Saponin (filtered) and 1% BSA in PBS] overnight at 4°C. Wells were washed three times with PBS, followed by 3×5 min washes with PBS, then covered with aluminum foil and incubated with secondary antibody diluted in ICC antibody diluent in RT. Three PBS wash and 3 × 5 min washes in PBS were repeated, and cells were stored in PBS with NucBlue Fixed Cell ReadyProbes Reagent (DAPI) (ThermoFisher, cat no: R37606) in 4°C until imaging. Confocal images were acquired using a Zeiss LSM 880 confocal microscope using the 63x oil objective.

Treated cultured fibroblasts were seeded at a concentration of 5 × 10⁴ cells/mL on Poly-L-Lysine-coated glass cover slips in a 12-well plate and allowed to grow overnight in growth media at 37 °C in a 5% CO₂ incubator. Cells were then fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.1% Triton X-100 and blocked after brief washings in 5% donkey serum at room temperature for 1 h. Cells were briefly washed and treated with primary antibodies VLCAD (1:1000, Vockley Lab) and HADHA (1:100, Santa Cruz Biotechnology, Dallas, TX, USA) overnight at 4 °C. After brief washing with 1 X tris buffered saline, pH 7.4 with Tween 20, cells were incubated with the secondary antibodies donkey anti-rabbit Alexa Fluor 488 and donkey anti-mouse Alexa Fluor 594 (1:1000, Invitrogen, Waltham, MA, USA) for 1 h at RT. Nuclei were counterestained with NucBlue Fixed Cell ReadyProbes Reagent (DAPI; Invitrogen). The cover slips were then mounted using mounting media before imaging. All images were taken on a Zeiss LSM710 Confocal microscope using 63× magnification. Images were analyzed using ImageJ [43 (link)].