Alexafluor 488 conjugated rabbit anti mouse secondary antibody

In paraffin-embedded kidney sections (3 µm), antigens were retrieved by the PTlink link system. After the slides were blocked with 10% BSA and 10% FBS for 1 h, they were incubated with anti-SOX9 (1/200; Millipore) or anti-type IV collagen (1:1000; Abcam) for 1 h, followed by an AlexaFluor^® 488 conjugated rabbit anti-mouse secondary antibody (1/200; Invitrogen); AlexaFluor^® 633 conjugated rabbit anti-mouse secondary antibody (1/200; Invitrogen) or AlexaFluor^® 568 conjugated rabbit anti-mouse secondary antibody (1/200; Invitrogen) for 1 h. The absence of a primary antibody was used as a negative control. Samples were mounted in prolong gold (Thermo Fisher Scientific; Waltham, MA, USA) and examined using a Leica DM-IRB confocal microscope.

For immunocytochemistry experiments, cells were grown on coverslips. After incubation, cells were fixed in paraformaldehyde 4% and permeabilized with 0.2% Triton-X100 for 10 min. After blocking with 3% BSA for 1 h, they were incubated with anti-SOX-9 ([1:100]; Millipore) overnight at 4 °C. Then, they were incubated for 1 h with the secondary antibody AlexaFluor^® 488 conjugated rabbit anti-mouse secondary antibody ([1/300]; Invitrogen; Waltham, MA, USA). Nuclei were stained with 4′,6-Diamidino-2-phenyindole (DAPI, Sigma-Aldrich; Saint Louis, MO, USA) as a control for equal cell density. The absence of a primary antibody was used as a negative control. Samples were mounted in Mowiol 40–88 (Sigma-Aldrich) and examined by a Leica TCS SP5 confocal microscope.

Freshly isolated human foreskin epidermis keratinocytes were suspended at a concentration of 10⁶ cells/ml in staining medium (DMEM containing 2% FBS and 10 mM HEPES), incubated with anti-ABCG2 antibody (clone 5D3, 1:25; BD Biosciences, Singapore) for 30 minutes on ice, washed with PBS containing 1% FBS, and then incubated with Alexafluor-488 conjugated rabbit anti-mouse secondary antibody (1:200; Gibco, Invitrogen) for 30 minutes, washed with PBS/1% FBS for 5 minutes three times, and resuspended in staining medium. The cells were kept on ice until flow cytometry analysis was performed. FACS was performed at the National University Medical Institute (Singapore) with a FACSadvantage SE (BD Biosciences, Mountain View, CA, USA) cell sorter.
To characterize the phenotype of ABCG2-positive and ABCG2-negative keratinocytes, the sorted cells were stained for 30 minutes at 4°C with anti-human antibodies against α6-integrin (GoH3), β1-integrin (4B7R), CD34 (8G12), CD71 (C2), keratin 14 (RCK107, phycoerythrin-conjugated, 1:100; all from BD Biosciences, USA), and p63 (4A4, 1:200; Santa Cruz Biotechnology Inc., Zoolem Marketing, Singapore).