Ab108985

Unless otherwise stated, all Western blotting was performed using equipment from BioRad as previously described [30 (link)]. Primary antibodies were used to detect BCRP (BXP-53, 1:5000; Enzo Life Sciences, Farmingdale, NY), β-actin (ab8227, 1:2000, Abcam, Cambridge, MA), transferrin receptor 1 (TFR-1, ab108985, 1:5000 Abcam), placental alkaline phosphatase (PLAP, ab133602, 1:10,000, Abcam), multidrug resistance-associated protein 1 (MRP1, ab3368, 1:2000, Abcam), cluster of differentiation 34 (CD34, ab81289 1:10,000, Abcam), histone H2A (25785, 1:1000, Cell Signaling, Danvers, MA), and glyceraldehyde 3-phosphate dehydrogenase (GAPDH, G8795, 1:1000, Sigma-Aldrich). HRP-linked secondary antibodies (anti-rabbit, anti-rat or anti-mouse, 1:2000 Sigma-Aldrich) were used to detect primary antibodies. After the addition of a Luminata Forte Western HRP substrate (Millipore, Billerica, MA), chemiluminescent protein-antibody complexes were visualized using a Fluorchem Imager (ProteinSimple, Santa Clara, CA). Semi-quantitative analysis of bands of the blots was performed using AlphaView Software (ProteinSimple).

For immunohistochemistry, tissue was embedded in paraffin and 5 μm thick sections prepared. After deparaffinization, tissue sections were quenched in 2% H₂O₂ (10 min, room temperature). Tissue sections were then blocked with an avidin/biotin blocking kit (Vector Laboratories, Burlingame, CA) followed by 5% serum corresponding to the source of the primary antibody. After 2 h at room temperature, tissues sections were incubated with primary antibodies to BCRP (BXP-21, 1:100; abcam), TFR-1 (ab108985, 1:200 Abcam), PLAP (ab133602, 1:1000, Abcam), or CD34 (ab81289 1:10,000, Abcam). After 16 h at 4°C, tissue sections were washed and incubated with biotinylated secondary antibodies for 60 min at room temperature (Vector Laboratories). Tissue sections were then stained using a 3,3′-diaminobenzidine peroxidase substrate kit (Vector Laboratories). After counterstaining with hematoxylin, tissue sections were dehydrated and imaged by light microscopy on a Olympus BX51 microscope (Waltham, MA) fitted with a ProgRes C14+ camera (Jenoptik, Jena, Germany). Negative controls for each secondary antibody are provided (Supplemental Fig 2).