Alexa fluor 488 conjugated anti goat

For immunofluorescence staining, HT-29 and HeLa cells were cultured on coverslips in 12-well plates. Post treatment with the indicated reagents, the cells were fixed with 4% paraformaldehyde for 30 min at RT. Anti-EGFR (Cell Signalling, 4267S) (1:50) and anti-TNFR1 (R&D systems, AD225) (1:50) or Anti-HA (Santa Cruz, sc805) (1:50) and anti-FLAG (Sigma Aldrich, F3165) (1:50) were incorporated to each sample in immunofluorescence blocking buffer (PBS with 3% BSA, 1% saponin and 1% Triton X-100) and then incubated at 4 °C overnight. Each sample was washed thrice with PBS. Alexa Fluor 488-conjugated anti-goat (Invitrogen, A-10055) (1:100) and Alexa Fluor 594-conjugated anti-rabbit (Invitrogen, A-21207) (1:100) or Alexa Fluor 488-conjugated anti-mouse (Invitrogen, A-11029) (1:100) and Alexa Fluor 594-conjugated anti-rabbit antibodies (Invitrogen, A-21207) (1:100) were incorporated into each sample in immunofluorescence blocking buffer and incubated at RT for 1 h. The nuclei were stained with DAPI for 5 min and evaluated using LSM980 (Carl Zeiss, Oberkochen, Germany) and ZEN (blue edition) programme.

Samples were treated with Histo VT One (Nacalai Tesque, Kyoto, Japan), blocked using 0.3% hydrogen peroxide and Blocking One Histo (Nacalai Tesque) to reduce nonspecific antibody interactions. The slides were incubated with monoclonal primary antibodies against human mitochondria (H-Mit) (Abcam, Cambridge, UK), Alb, αFP, IGF, CPS-1, and HNF4α at 4°C overnight. After washing, specimens were treated with Mouse on Mouse ImmPRESS Horseradish Peroxidase (Vector Laboratories, Burlingame, CA, USA) for 1 h at room temperature and then reacted with diaminobenzidine horseradish peroxidase substrate (Vector Laboratories). Finally, the slides were visualized under a microscope (FSX 100, Olympus, Tokyo, Japan).
To confirm cell distribution in the liver after transplantation, double fluorescence staining of human Alb and H-Mit was performed. The samples were incubated overnight at 4°C with a goat antibody against human Alb (Abcam) and a mouse antibody against H-Mit; Alexa Fluor 488-conjugated anti-goat (Invitrogen) and Alexa Fluor 568-conjugated anti-mouse were used as secondary antibodies, respectively. Stained slides were analyzed using a laser fluorescence microscope.