α aldh1

For ALDH1/Hypoxyprobe and β-catenin/Hypoxyprobe double labeling, mice were injected intravenously with 60 mg/kg of the pimonidazole solution, 90 min later, the mice were euthanatized and tumor tissues were removed and snap-frozen. Frozen tissue sections were then interrogated with FITC-conjugated α-pimonidazole (Hypoxyprobe Inc., USA) and α-ALDH1 (Abcam, USA)/β-catenin (Cell Signaling Technology, USA) followed by respective Cy3-conjugated secondary IgG. Coverslips were then mounted with DAPI stain solution. For Periodic Acid Schiff (PAS)-CD31 double IHC staining, paraffin sections were cut into 5 μm sections and fixed in 4% paraformaldehyde. The sections were firstly incubated with α-CD31 (Cell Signaling Technology, USA), then exposed to 1% sodium periodate and incubated with PAS (BestBio, Shanghai, China).

For ALDH1/Hypoxyprobe and β-catenin/Hypoxyprobe double labeling, mice were injected intravenously with 60 mg/kg of the pimonidazole solution, 90 min later, the mice were euthanatized and tumor tissues were removed and snap-frozen. Frozen tissue sections were then interrogated with FITC-conjugated αpimonidazole (Hypoxyprobe Inc., USA) and α-ALDH1 (Abcam, USA)/β-catenin (Cell Signaling Technology, USA) followed by respective Cy3-conjugated secondary IgG. Coverslips were then mounted with DAPI stain solution. For Periodic Acid Schiff (PAS)-CD31 double IHC staining, para n sections were cut into 5 μm sections and xed in 4% paraformaldehyde. The sections were rstly incubated with α-CD31 (Cell Signaling Technology, USA), then exposed to 1% sodium periodate and incubated with PAS (BestBio, Shanghai, China).
Secondary nude mouse xenograft model MDA-MB-231/MDA-MB-468 tumors tissues were minced into small pieces and processed with collagenase for 2 h at 37°C. After centrifugation, the cell pellets were trypsinized and passed through an 80-μm lter to produce single-cell suspension, then living cells were sorted out by uorescence-activated cell sorting. Each nude mouse was inoculated with 1 × 10 4 cells from control/Bevacizumabtreated/Bevacizumab + SHH002-hu1-treated tumors in one of the inguinal mammary fat pads. The growth of tumors was monitored and tumor sizes were measured weekly.