Rabbit anti afp

Cells were fixed in 4% formaldehyde (Sigma-Aldrich) for 30 minutes at RT, rinsed three times in PBS containing 0.1% Tween 20 (PBST) for 10 minutes, permeabilized in PBS containing 0.1% Triton X-100 (Sigma-Aldrich) for 15 minutes, and blocked for 1 hour in PBS containing 5% normal goat serum (Jackson ImmunoResearch, West Grove, PA, USA). Cells were incubated overnight at 4°C with the following primary antibodies diluted in PBS containing 5% normal goat serum: rabbit anti-ALB (1:50; Dako, Glostrup, Denmark), rabbit anti-AFP (1:200; Dako); rabbit anti-AAT (1:200; Abcam), mouse anti-HNF4A (1:200; Abcam). Cells were rinsed six times in PBST for 10 minutes each. Thereafter, cells were incubated for 1 hour at RT with appropriate secondary antibodies diluted in PBST: Alexa Fluor 488 or 594 goat anti-rabbit IgG and Alexa Fluor 594 goat anti-mouse IgG (1:200; Invitrogen). Cells were washed six times in PBST, and mounted in 4'-6-diamidino-2-phenylindole (DAPI, Sigma-Aldrich).

Cells were fixed in 4% paraformaldehyde (Nacalai Tesque) in PBS, permeabilized with 0.1% Triton X-100 (Nacalai Tesque), and were then blocked with 20% Blocking One (Nacalai Tesque, Japan) in PBST (0.1% Tween-20 in PBS). Antibodies were diluted in 20% Blocking One (Nacalai Tesque, Japan) in PBST (0.1% Tween-20 in PBS). Cells were counterstained with 6-diamidino-2-phenylindole (DAPI) (Roche Diagnostics, Switzerland).
The following antibodies were used: rabbit anti-AFP (Dako, Glostrup, Denmark), goat anti-ALB (Bethyl), mouse anti-OCT3/4 (Santa Cruz), goat anti-SOX17 (R&D Systems), and Alexa 568-conjugated and Alexa 488-conjugated antibodies (Invitrogen). Positive cells versus total cells (DAPI-positive cells) were quantified using the Metamorph Image Analysis Software (Molecular Devices).