Cells were seeded into glass Lab-Tek Chamber Slides (8 wells; 0.8cm2/well) at a density of 15–20 × 103/well, allowed to attach for 24 h at 37 °C. For P-gp detection AS cells were previously treated with 1 µg/ml docetaxel, 150 µM propranolol and their combination. Once the confluence was reached the cells were fixed for 10 min in acetone at RT and treated with a blocking solution. Then, the slides were incubated with diluted primary antibody anti-β1 Adrenergic Receptor (Abcam), anti-β2 Adrenergic Receptor (Abcam), anti-β3 Adrenergic Receptor (Abcam) and anti-P-gp (JSB-1, Abcam) in 1% BSA in PBS for 1 h at RT. After 3 wash steps with PBS, cells were then incubated with EnVision + System-HRP Labelled polymer secondary antibody (Dako, Denmark). Color development was obtained with AEC ready to use solution (Dako, Denmark), whereas the nuclei were counterstained with hematoxylin. Then, the slides were sealed with Kaiser mounting medium for optical observation.
For the evaluation of CD-31 and CD-34,the slides were incubated with diluted primary antibody anti-CD-31 (PECAM-1, Novocastra) and anti-CD-34 (clone QBEND-10, Novocastra) in 3% BSA in PBS for 1 h and revealed by secondary antibody FITC-Goat anti-mouse Ig (BD Biosciences). Thereafter the slides were sealed with Vectashield (Vector Laboratories) for fluorescence examination by means of DMi8 Leica microscope.
For the evaluation of CD-31 and CD-34,the slides were incubated with diluted primary antibody anti-CD-31 (PECAM-1, Novocastra) and anti-CD-34 (clone QBEND-10, Novocastra) in 3% BSA in PBS for 1 h and revealed by secondary antibody FITC-Goat anti-mouse Ig (BD Biosciences). Thereafter the slides were sealed with Vectashield (Vector Laboratories) for fluorescence examination by means of DMi8 Leica microscope.