Ab47709

Paraffin-embedded tumor tissues were sliced into 5-μm thick sections and mounted on glass. Slides were deparaffinized and rehydrated in 10 min through a graded alcohol series to deionized water in 1% Antigen Unmasking Solution (Vector Laboratories, Burlingame, CA, USA) and microwaved to enhance antigen retrieval. Tissue samples were sequentially incubated with anti-mouse immunoglobulin coupled to horseradish peroxidase (HRP). Slides were incubated with the specific primary anti-GST-π (ab47709; Abcam, Cambridge, UK) and anti-P-gp (P7965; Sigma, St. Louis, MO, USA) monoclonal antibodies with an HRP-conjugated secondary antibody (A1293; Sigma), and then stained with 3,3-diaminobenzidine and counterstained with hematoxylin and eosin. In addition, 10 tissue samples from patients with chronic cholecystitis obtained by cholecystectomy were used as the control group. Two pathologists independently observed and interpreted the results of the immunohistochemical staining.

The assay was implemented in line with the previous description [23 (link)]. Generally, the separated proteins were and transferred onto polyvinylidene fluoride (PVDF) membranes (Millipore, Bedford, MA, U.S.A.). Then, the membranes were blocked with 5% non-fat milk. Afterwards, the membranes were incubated with primary antibodies, including ZEB1 (1:1000, ab181451; Abcam, Cambridge, MA, U.S.A.), P-gp (1:1000, ab3364; Abcam) and GST-π (1:800, ab47709; Abcam) and GAPDH (1:8000, ab8245, Abcam), followed by probed with corresponding secondary antibody (Abcam) to combine these primary antibodies. At last, proteins blots were assessed with an enhanced chemiluminescence detection kit (Millipore).