Diaminobenzidine colorimetric reagent solution

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)].

Six-week-old female BALB/cA nu/nu mice were purchased from Vital River Laboratory Animal Technology Company (Beijing, China) and maintained in an Animal Biosafety Level 3 Laboratory at the Animal Experimental Center of Wuhan University. All animal experiments were performed according to the Wuhan University Animal Care Facility and National Institutes of Health guidelines. Approximately 3 × 10⁶ SGC7901 cells and 1 × 10⁶ HELF^FAP cells (Group I, n = 5), 3 × 10⁶ SGC7901 cells and 1 × 10⁶ HELF^NC cells (Group II, n = 5) were harvested and suspended in 200 ml of PBS and Matrigel (BD Bio-science, USA) (1:1) and injected subcutaneously into the right flank of each mouse. The size of subcutaneous tumors was recorded every two days. Five weeks later, mice were sacrificed, and the tumors were removed. The weight of tumors was recorded and statistically analyzed. The xenograft tumor slides were incubated with the following primary antibodies: anti-CD31 was purchased from ABclonal (Boston, USA) and anti-Ki67 from Cell Signaling Technology (Boston, USA). Anti-rabbit or anti-mouse peroxidaseconjugated secondary antibody (ABclonal, Boston, USA) and diaminobenzidine colorimetric reagent solution (Dako, Carpinteria, CA) were used. The staining processes were performed according to standard methods.