Alexa 488 green

pTriAR plasmid expressing full length AR was provided by Taiho Pharmaceutical. DU145 Cells were transfected with pTriAR plasmid using FuGENE HD transfection reagent according to the manufacturer’s protocol.
LNCaP and DU145 Cells with stably transfected AR were grown on glass coverslips, then fixed with cold acetone for 2 min at room temperature. Following fixation, cells were blocked with FBS for 20 min and incubated with AR antibodies (sc816, Santa Cruz) for 60 min. The appropriate secondary antibodies conjugated with Alexa 488 (green) (Cell Signaling Technology, Beverly, MA, USA) were added and allowed to incubate for 45 min. Cells were counterstained with propidium iodide for 10 min and visualized by fluorescence microscopy.

For IF staining, cells were grown on glass coverslips, then fixed with methanol for 5 min at room temperature. Following fixation, cells were blocked with Dako buffer (S0809, Agilent) for 1 h, and incubated with primary antibodies overnight at 4 °C, then with appropriate secondary antibodies conjugated with Alexa 555 (red) or Alexa 488 (green) (Cell Signaling Technology). Cells were counterstained with DAPI (DUO82040, Sigma-Aldrich) for 10 min and visualized by fluorescence microscopy (Eclipse-NiE NIKON microscope).
Xenografts were collected and fixed in 4% PFA prior to paraffin embedding, sectioning, staining with hematoxylin and eosin, and with immunostaining conducted as described previously [23 (link)]. Images were acquired on a ZEISS Axioscope 5 microscope.

For IF staining, cells were grown on glass coverslips and then fixed with 4% paraformaldehyde for 15 min at room temperature. Following fixation, the cells were permeabilized using 0.5% Triton X-100 for 15 min and blocked with 5% goat serum for 1 h. Then, the cells were incubated with primary antibodies overnight at 4 °C and appropriate secondary antibodies conjugated with Alexa 555 (red) or Alexa 488 (green) (Cell Signaling Technology). DAPI-containing mounting medium (Abcam, Shanghai, China) was used to stain nuclei and mount cells. A Leica SP5 Laser Scanning Confocal Microscope (Leica Microsystems, Buffalo Grove, IL, USA) was used to capture and analyze images.