IHC analysis staining was performed as previously described 23 (link). Antibodies against ITGA5 (diluted 1:50), EFNB2 (diluted 1:200), p-S6 (diluted 1:75), Ki-67 (diluted 1:100) and CD31 (diluted 1:100) were used. A modified histologic score (H-scores, [{% of weak staining} × 1] + [{% of moderate staining} × 2] + [{% of strong staining} × 3]) was used to evaluate IHC staining 24 (link), 25 (link). Each staining obtained an H-score between 0 and 300, and the average of H-score for all the cases was calculated.
For IF assays, Cells were treated with DMSO, Rapa (20 nM), RAD001 (50 nM) or MHY1485 (10 µM) for 24 h and then stained as previously described 23 (link). Primary antibodies against ITGA5 (diluted 1:50), EFNB2 (diluted 1:200), or CD44 (diluted 1:1000) and FITC-conjugated secondary antibody (diluted 1:1000) were used. DAPI (Beyotime) was used to stain nuclei. The images were captured by LSM880 + Airyscan confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
For IF assays, Cells were treated with DMSO, Rapa (20 nM), RAD001 (50 nM) or MHY1485 (10 µM) for 24 h and then stained as previously described 23 (link). Primary antibodies against ITGA5 (diluted 1:50), EFNB2 (diluted 1:200), or CD44 (diluted 1:1000) and FITC-conjugated secondary antibody (diluted 1:1000) were used. DAPI (Beyotime) was used to stain nuclei. The images were captured by LSM880 + Airyscan confocal laser scanning microscope (Carl Zeiss, Oberkochen, Germany).
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