Alexafluor 488 conjugated secondary antibody

For immunostaining of cilia in zebrafish embryos, embryos were fixed in 4% paraformaldehyde at 4 °C overnight and blocked with blocking solution (1 × PBS, 3% BSA, and 1% Triton X-100) at RT for 2 h. Zebrafish embryos were incubated with anti-acetylated-α-tubulin (Proteintech, CL594-66200, 1:500 dilution) at 4 °C overnight. Next, embryos were washed PBST (with 1% Triton X-100) for 30 min and incubated with Alexa Fluor 488-conjugated secondary antibodies (Sigma, 1:500 dilution) at 4 °C overnight. The nuclei of the fixed embryos were stained with 4', 6'-diamidino-2-phenylidole (DAPI). Images were acquired using a confocal microscope (LSM700, Zeiss, Oberkochen, Germany) at the Chronic and Metabolic Diseases Research Center of Sookmyung Women’s University.

To evaluate cell proliferation, 5 × 10⁵ cells were grown on sterile glass coverslips, rinsed and fixed with 4% w/v paraformaldehyde for 15 min, then permeabilized with 0.1% v/v Triton-X100 for 5 min on ice, washed three times with PBS and stained with an anti-Ki67 antibody (Abcam) for 1 h at room temperature. After washing, samples were incubated with an AlexaFluor 488-conjugated secondary antibody (Millipore) for 1 h and re-washed. Finally, cells were stained with PI (1 μg/ml) to counterstain the nuclei, and washed again. The coverslips were mounted with 4 μL of Gel Mount Aqueous Mounting and examined by confocal microscopy as detailed above. Early and late apoptosis was measured by the Annexin V/Propidium Iodide Apoptosis Detection Kit (Sigma Chemical Co.). 1 × 10⁵ cells were analyzed with a FACS-Calibur flow cytometer (Becton Dickinson). The percentage of cells positive to annexin V-FITC and PI was calculated with the Cell Quest software (Becton Dickinson). Cell senescence was evaluated on 5 × 10⁵ cells fixed and stained with the Senescence Cells Histochemical Staining Kit (Sigma Chemical Co.), following the manufacturer's instruction. Samples were examined with a Leica DC100 fluorescence microscope (Leica Microsystems GmbH, Wetzlar, Germany). For each experimental point, a minimum of five microscopic fields were examined.

Human melanoma A375 cells (ATCC, Manassas, VA, USA) were maintained in DMEM medium supplemented with 10% v/v fetal bovine serum, 1% v/v penicillin-streptomycin, 1% v/vl-glutamine. Cells were maintained in a humidified atmosphere at 37 °C, 5% CO₂. To measure the expression of ABC transporters, 1 × 10⁶ cells were rinsed and fixed with 2% w/v paraformaldehyde (PFA) for 2 min, washed three times with PBS and stained with anti-P-glycoprotein (Pgp/ABCB1) (Kamiya, Hamamatsu City, Japan), anti-MDR-related protein 1 (MRP1/ABCC1) (Abcam, Cambridge, UK) and anti-breast cancer resistance protein (BCRP/ABCG2) (SantaCruz Biotechnology Inc., Santa Cruz, CA, USA) antibodies for 1 h on ice, followed by an AlexaFluor 488-conjugated secondary antibody (Millipore, Billerica, MA, USA) for 30 min. One-hundred-thousand cells were analyzed with EasyCyte Guava™ flow cytometer (Millipore), equipped with the InCyte software (Millipore). Control experiments included incubation with non-immune isotype antibody.

5 × 10⁵ cells were grown on sterile glass coverslips, rinsed and fixed with 4% w/v paraformaldehyde for 15 min. To visualize surface CAXII and Pgp, the samples were washed with PBS and stained with an anti-CAXII antibody or an anti-Pgp antibody conjugated to phycoerythrin (Millipore) for 1 h. After washing, samples stained for CAXII were incubated with an AlexaFluor 488-conjugated secondary antibody (Millipore) for 1 h and re-washed. The coverslips were mounted with 4 μL of Gel Mount Aqueous Mounting and examined with an Olympus FV300 laser scanning confocal microscope (Olympus Biosystems, Tokyo, Japan). For each experimental point, a minimum of five microscopic fields were examined.

1 × 10⁶ cells, rinsed and fixed with 2% w/v paraformaldehyde for 2 min, were washed three times with PBS and stained with an anti-CRT antibody (Affinity Bioreagents) for 1 h on ice. After washing, samples were incubated with an AlexaFluor 488-conjugated secondary antibody (Millipore) for 30 min and re-washed. Samples were analyzed with a Guava easyCyte flow cytometer (Millipore). For each analysis 10,000 events were collected. Control experiments included incubation with non immune isotypic antibody followed by the secondary antibody. The results were analyzed with the easyCyte software (Millipore).