Alexa 594

Cells were fixed with 4% paraformaldehyde (PFA) for 20 min and blocked with blocking buffer (5% NGS, 0.3% TritonX100 in PBS) for 1 hr at room temperature. Cells were subsequently incubated with anti-Rictor and anti-ABLIM1 antibodies at 4°C overnight. After washing with PBS three times, cells were stained with secondary antibodies conjugated to Alexa488 (ZSGB-BIO), Alexa594 (ZSGB-BIO) or Alexa594-labeled phalloidin (Invitrogen) at 37°C for 1 hr. Images were obtained by confocal microscopy (OLYMPUS) and further processed and analyzed with ImageJ software.

Immunofluorescence analysis was performed after 24 h cell culture. Cells were washed 3 times in PBS containing 1‰ tween-20 for 5min, and fixed in 4% paraformaldehyde for 20 min and permeabilized in 0.5% Triton X-100 for 20 min at room temperature. Cells were washed 3 times and blocked in 1% BSA for one hour. Subsequently, cells were incubated with primary antibody overnight at 4^°C. After washing, cells were incubated with secondary anti-rabbit IgG antibody conjugated with Alexa 594 (ZSGB-BIO, China) for 30 min at 37°C. Nuclei were stained with 1μg/mL DAPI (4′,6-diamidino-2-phenylindole) for 5 min. Finally, cells were visualized under a laser-scanning confocal microscope (Nikon Ti-E, Japan) for image acquisition. The experiments were repeated in duplicate.

Paraffin sections of Matrigel plugs were stained with primary antibody against CD31 (1:50, Abcam ab28364) at 4 °C overnight. Subsequently, the slides were incubated with goat-anti-rabbit antibody conjugated Alexa 594 (ZSGB-BIO, Beijing, China) for 1 h at room temperature. Nuclei were detected with DAPI. Slides were photographed by an FV-1000 laser scanning confocal biological microscope (Olympus).