Methods of P-glyp Expression Using IHC Technique

The CD243/glycoprotein P monoclonal antibody (C219) from Thermo Scientific Pierce Antibodies is a mouse monoclonal antibody to C219 to ABCB1, ATP-binding cassette, sub-family B (MDR/TAP), member 1A, Abcb1a, Mdr1a. This antibody recognizes rat, mouse, hamster, and human antigen. The formalin-fixed paraffin extracted tissue blocks were sectioned at 3–4 μm, using a microtome (Leica, Germany), mounted on tissue bond-coated slides (Biocare, USA). Immunohistochemistry was done by the standard protocol. The tissue section on coated slides was fixed overnight at 60 °C in a dry oven, deparaffinized in xylene and rehydrated through graded ethanol series. Sections were blocked with 3% hydrogen peroxide in methanol for 30 min to quench any endogenous peroxidase activity, if present, and were then processed for antigen retrieval in Pascal (DAKO Cytomation, California) by placing in sodium citrate buffer (pH 6.0). Sections were incubated for an hour with P-glycoprotein antibody (C219), followed by treatment with polymer-based secondary antibody kit (Dakopatts, Envision kit, Denmark). Bound antibody was visualized using diaminobenzidine. Sections were counter-stained with hematoxylin and mounted. Positive and negative (by omitting primary antibody) controls were run with all batches.