3 3 diaminobenzidine tetrahydrochloride

The tissue sections were initially deparaffinized in an oven at 65°C for 2 h, immersed in xylene 3 times and subsequently heated to 100°C in 10 mM citrate buffer (Origene Technologies, Inc., Beijing, China), pH 6.0, for 30 min. Sections were blocked with 1% bovine serum albumin (A1933; Sigma-Aldrich, Merck KGaA, Darmstadt, Germany) and 5% normal goat serum (S-1000; Vector Laboratories, Inc., Burlingame, CA, USA) in PBS plus 0.04% tween 20 at room temperature for 1 h. Sections were subsequently incubated with primary anti-LGR5 (1:100; ab75850; Abcam, Cambridge, UK), anti-GATA6 (1:150; sc-9055; Santa Cruz Biotechnology, Inc., Dallas, TX, USA) and anti-β-catenin antibodies (1:200; 51067-2-AP; ProteinTech Group, Inc., Chicago, IL, USA) at 4°C overnight. Samples were treated with a 2-step plus Poly-HRP anti-mouse/rabbit IgG detection system (cat. no. PV-9000; Origene Technologies, Inc.) and 3,3′-diaminobenzidine tetrahydrochloride (cat. no. ZLI-9032; Origene Technologies, Inc.), according to manufacturer's protocols. Finally, samples were counterstained with Mayer's hematoxylin (cat. no. MHS16; Sigma-Aldrich; Merck KGaA) at room temperature for 5 min and mounted with Histomount mounting medium (HS-103; National Diagnostics, Atlanta, GA, USA).

IHC was performed on paraformaldehyde-fixed paraffin sections. The SP link IHC Detection kit (Biotin-Streptavidin HRP Detection systems; catalog no. SP-9001) was purchased from OriGene Technologies, Inc. (Beijing, China). In brief, rabbit anti-human polyclonal Glut1 (catalog no. sc-7903; dilution, 1:100), rabbit anti-human polyclonal MMP2 (catalog no. sc-10736; dilution, 1:100) and rabbit anti-human polyclonal MMP9 (catalog no. sc-10737; dilution, 1:100) antibodies were purchased from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA) and used to detect the protein expression in IDBC tissues. Tissue sections were incubated with primary antibodies at 4°C for 24 h. Goat anti-rabbit secondary antibodies from the SP link IHC Detection kit were added to the sections and tissue sections were incubated at 37°C for 1 h. Protein expression was visualized using 3,3-diaminobenzidine tetrahydrochloride (OriGene Technologies, Inc.) and observed through a BX46 upright microscope (Olympus Corporation, Tokyo, Japan). The staining results for the Glut1, MMP2 and MMP9 proteins were semi-quantitatively calculated by multiplying the staining intensity and the percentage of positive normal cells, as previously reported (16 (link)).