Bxp 21

Each population of sorted ABCG2-positive, ABCG2-negative keratinocytes and unsorted keratinocytes from human epidermis were collected, centrifuged at 4°C, and the cell pellets were homogenized in RIPA buffer (1% Nonidet P-40, 0.5% sodium deoxycholate, 150 mM sodium chloride, 1 mM phenylmethylsulfonyl fluoride and 1 μg/ml leupeptin) at 4°C. The cell protein lysates was quantified using a BCA kit (Pierce, Rockford, IL, USA). An equal amount of protein was electrophoresed in a 7.5% SDS-polyacrylamide gel, transferred onto a nitrocellulose membrane. The membrane was blocked with 2% bovine serum albumin in Tris-buffered saline for 3 hours, washed with Tris-buffered saline for 1 hour, and incubated with anti-ABCG2 antibody (BXP-21, 1:100; Millipore) diluted in Tris-buffered saline with 0.1% Tween-20 overnight at 4°C. After washing, anti-mouse IgG (horseradish peroxidase-conjugated, 1:2,000; Santa Cruz Technologies, Santa Cruz, Biotechnologies Inc., Zoolem Marketing, Singapore) was applied for 1 hour at room temperature. Finally, chemiluminescence was carried with the Supersignal chemiluminescent substrate (Pierce).

After culture with DEX or cortisol, explants were processed for immunohistochemical analysis as previously described.3, 19 Slides were incubated (overnight at 4°C) with primary antibodies: anti‐mouse BCRP (1:200, BXP‐21, Millipore, Billerica, MA), and anti‐mouse cytokeratin 7 (CK7, 1:500, Dako). Mouse or rabbit IgG1 (Dako) was added instead of primary antibody in controls. After incubation, slides were washed and incubated with the corresponding biotinylated secondary antibody (1:300, 1 hour, Dako). Sections were then washed in PBS (3x for 10′ each time) and incubated with streptavidin‐HRP (30 minutes; Dako). Chromogenic detection of horseradish peroxidase (HRP) activity was achieved by 3,3′‐diaminobenzidine (DAB) reagent (DAB peroxidase substrate kit, Dako). Slides were counterstained with haematoxylin, dehydrated in ascending grades of ethanol and cover slipped. Slides were visualized with an Olympus BX61 upright, motorized microscope with an Olympus DP72 digital camera (Olympus, Tokyo, Japan).