Immunocytochemistry/Immunofluorescence Protocol for CHREBP Antibody (NB400-136)

Procedure for immunofluorescent staining

1. Wash human hepatocytes grown on cover-slips with PBS twice.
2. Fix cells with 10% buffered formalin for 25 minutes at room temperature.
3. Wash cells with PBS 3 times.
4. Incubate with 0.5% Triton X-100 in PBS for 5 minutes.
5. Wash cells with PBS 3 times.
6. Block with 1% BSA in PBS for 15 minutes at 37C.
7. Incubate with rabbit anti-ChREBP antibody (NB 400-136) 1:100-1:500 in PBS with 1% BSA for 30 minutes at 37C.
8. Wash cells with PBS 3 times.
9. Incubate with Goat anti-rabbit Alexa Fluor 568 antibody 1:2,000 in PBS with 1% BSA for 30 minutes at 37C.
10. Wash cells with PBS 3 times.
11. Mount by Vectashield mounting medium with DAPI.
12. Take pictures by using Rhodamine and DAPI filters.