Goat anti shh

Elution of AnxA2 (26 (link))and Western blot with rabbit anti-AnxA2, mouse anti-P-Y23-AnxA2, mouse anti-beta-actin, rabbit anti-HGF (all from Santa Cruz Biotechnology), rabbit anti-IGF-1(Abcam) or goat anti-Shh (R&D Systems) antibodies were described previously (12 (link)) Immunohistochemistry (IHC) staining for E-Cadherin and HGF was performed with rabbit anti-E-Cadherin (Abcam) and rabbit anti-HGF (Santa Cruz Biotechnology) antibodies using a standard protocol on an automated stainer from Leica Microsystems. IHC for SMA was performed as previously described (27 (link)). IHC for AnxA2 was conducted with mouse anti-AnxA2(Invitrogen), or mouse anti-P-Y23-AnxA2 antibodies, IHC for Shh with goat-anti-Shh (R&D), and IHC for IGF-1 with rabbit-anti-IGF-1(Abcam) antibodies as described previously (13 (link)). All IHC slides were analyzed and scored by a pathologist (A.L). The levels of secreted IGF-1 were measured by ELISA (R&D) following the manufacturer’s instructions.

Immunohistochemistry (IHC) staining for Shh, c-Met, E-Cadherin, IGF-1 and Ki67 were performed by hand. Tissue blocks with poor quality were excluded from the study. The slides for all stainings were hydrated; antigen retrieval was performed in a pressure cooker with citrate buffer (pH 6.0) for Shh and Ki67, in a steamer with citrate buffer (pH 6.0) for c-Met and IGF-1 and with Tris-EDTA buffer (pH 9.0) for E-Cadherin. Then, the slides were blocked in peroxidase, avidin and biotin block sequentially. Goat-anti-Shh (R&D), rabbit-anti-c-Met (Santa Cruz), rabbit anti-IGF-1 (Abcam), rabbit anti-E-Cadherin (Abcam), or rabbit anti-Ki67 (Abcam) primary antibodies at 1:50 (Shh, c-Met, E-Cadherin), 1: 500 (Ki67) and 4µg/ml (IGF-1) were added, and the slides were incubated for 1 hour at room temperature. Then, rabbit anti-goat or goat anti-rabbit biotinylated secondary antibodies (Vector Laboratories) respectively, were added for 30 minutes at room temperature. The signal was amplified and detected using the ABC Vectastain kit (Vector Laboratories) according to the manufacturer’s instructions. The slides were developed using DAB and counterstained by hematoxylin. Lastly, the slides were dehydrated and mounted. All IHC slides were analyzed and scored by a pathologist. Immunofluorescence staining of c-Met and Shh was described in Supplementary Materials and Methods.