594 anti rabbit

Liver tissue was analyzed through cryostat-sectioned staining and confocal microscopy. Liver tissue was dissected in Krebs buffer (125.35 mmol/L NaCl, 5.9 mmol/L KCl, 1.2 mmol/L NaHPO₄, 15.5 mmol/L NaHCO₃, 1.2 mmol/L MgCl₂, 11.5 mmol/L D-glucose, and 2.5 mmol/L CaCl₂). Liver tissue was fixed in 4% paraformaldehyde at 4 °C for 20 min, followed by overnight incubation in 1 X Tris-buffered saline (TBS) at 4 °C. Dehydration was performed in 20% sucrose in TBS at 4 °C. Tissue was trimmed and placed in 1:1 optimum cutting temperature (OCT)/20% sucrose in TBS and frozen by liquid nitrogen. Then, 8 mm-thickness cryostat sections were prepared on slides and used for immunohistochemistry. Cryostat sections were blocked with 0.5% Triton X-114, 4% skim milk in TBS for 1h at room temperature, rinsed with TBS twice for 10 min each, and then incubated with anti-alpha fetoprotein (rabbit, 1:100, Proteintech, Rosemont, IL, USA) for 48 h on a rocker at 4 °C. The slides were rinsed with TBS twice for 10 min each and then incubated with 594-anti-rabbit (Jackson ImmunoResearch, West Grove, PA, USA) for 2 h at room temperature. The slides were rinsed with 1× TBS three times for 10 min each, dried, and mounted (mounting medium with 4′,6-diamidino-2-phenylindole (DAPI). An Olympus Fluoview FV1000 confocal laser scanning microscope (Olympus, Shinjuku, Tokyo, Japan) was used for all imaging analyses.