Ls b5729

Cells were seeded on chamber slides and treated with either 5 μM azacytidine or 200 μM zebularine for 72 h. After washing with PBS, cells were fixed with acetone-methanol (1:1) for 20 min at −20°C. Then cells were blocked with 5% BSA for 1 h, followed by incubation with anti-ABCB4 antibody (LifeSpan LS-B5729, 1:200) at 4°C overnight. Finally, cells were incubated with Alexa Fluor 488-conjugated secondary antibody, nuclei were stained with DAPI and mounted with fluorescent mounting media (Dako Cytomation, Glostrup, Denmark).

Lung cancer tissue array were purchased from Pantomics, Inc. (Richmond, CA, USA). In brief, slides were treated with high pH antigen retrieval buffer. Rabbit anti-human ABCB4 (1:200; LS-B5729, lifespan biosciences, USA) and normal rabbit serum were used for stainings. Slides were incubated with ABCB4 antibody at 4°C overnight. After extensive washing, sections were incubate with secondary antibody, followed by staining with vector DAB (Vector Laboratories). The slides were washed and counterstained with hematoxylin for 5 min. All slides were analyzed under the Hamamatsu NDP slide scanner (Hamamatsu Nanozoomer 2.0HT) and its viewing platform (NDP. Viewer).