Image scop software

The xenograft tumor slides were incubated with the following primary antibodies: phospho-AMPK (Thr172) (1:100), phospho-ACC (Ser79) (1:100) (Cell Signaling Technology, USA). Anti-rabbit peroxidase-conjugated secondary antibody (Santa Cruz Biotechnology) and diaminobenzidine colorimetric reagent solution purchased from Dako (Carpinteria, CA) was used. The staining process were according to standard methods. Assessment of the staining was performed using the Image-scop software (Media Cybernetics, Inc.) according to the staining intensities and the percentage of positively stained cells, as described [53 (link)].

For immunohistochemistry, the slides were incubated with primary antibodies referred above, which was followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Finally, the staining processes were performed with diaminobenzidine colorimetric reagent solution (Dako, Carpinteria, USA) and hematoxylin (Sigma Chemical Co., USA). For in situ hybridization analysis, hsa-miR-1247-3p miRCURY LNA detection probe (Exiqon, Denmark) was used and the total staining processes were carried out according to manufacturer’s protocols described before^{47 (link)}. Images were captured with Aperio ScanScope AT Turbo (Aperio, USA) and assessed with image-scop software (Media Cybernetics, Inc.)

For immunohistochemistry, the slides were incubated with above-mentioned primary antibodies, followed by incubation with horseradish peroxidase-conjugated secondary antibodies (Santa Cruz Biotechnology). Finally, the staining processes were performed with diaminobenzidine colorimetric reagent solution (Dako, Carpinteria, USA) and hematoxylin (Sigma Chemical Co., USA). For in situ hybridization analysis, hsa-miRNA-21 miRCURY LNA detection probe (Exiqon, Denmark) was used, and the total staining processes were carried out according to manufacturer’s protocols. Images were captured with Aperio ScanScope AT Turbo (Aperio, USA) and assessed with image-scop software (Media Cybernetics, Inc.).