Alexa 555 red

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.