Ab3380

Protein extracts of mouse ventricles or fibroblasts were prepared in RIPA buffer containing protease-inhibitor and phosphatase-inhibitor tablets (Roche, Vienna, Austria). After mechanical disruption of tissue samples, equivalent amounts of protein were separated on a SDS polyacrylamide gel, followed by electro-transfer to nitrocellulose membrane. Nonspecific antibody binding was blocked by incubation in 5% (m/v) non-fat dry milk powder in TBS-T (20 mM Tris-Cl, pH 7.5, 150 mMNaCl, 0.1% (v/v) Tween 20) at room temperature for 1 h. Then, samples were incubated overnight at 4°C with antibodies: anti-ABCG2 (#ab3380, Abcam) and anti-α-tubulin (#ab4074, Abcam) for ventricular tissues, and anti-ABCG2 (#AV43649, Sigma-Aldrich, Saint Louis, USA) and anti-GAPDH (#sc-25578, Santa Cruz Biotechnology, Heidelberg, Germany) for fibroblasts. After 1 h incubation in room temperature with peroxidase-labeled secondary antibody (Pierce Biotechnology, Rockford, USA), specific immunoreactive signals were detected using Enhanced ChemiLuminescence kit (Amersham Biosciences, Buckinghamshire, UK) or West Pico Substrate (Thermo Scientific, Rockford, USA).

IHC for BCRP was performed on the paraffin-embedded FM and placenta blocks prepared as above. Placental tissue served as a positive control as BCRP is known to be expressed in the placenta [10 (link)]. FMs without the application of the BCRP primary antibody served as the negative control.
Five-µm tissue sections were cut and adhered to slides with a positive charge. Deparaffinization was performed with Xylene. Sections were rehydrated with 100% ethanol, 95% ethanol, and 70% ethanol. An IHC kit from Abcam (ab64264) was used and the manufacturer’s instructions were followed. An antigen retrieval system was used for epitope unmasking utilizing the Tris-EDTA buffer (pH = 9.0). Placenta and FM sections were incubated with a BCRP primary antibody (Abcam [ab3380]; 1:1000 dilution) at 4 °C overnight for immunostaining. For the negative control, FM sections were incubated overnight in 3% BSA TBS-T without a primary antibody. An IHC antirabbit antibody (1:500, Vector Laboratories, CA, USA) was added for 10 minutes at room temperature, followed by DAB as a chromogen and hematoxylin as a counter stain for color development. Slides were examined with bright field microscopy for the presence and localization of BCRP. Images were taken at 20× and 40× magnification.