Rabbit anti c met

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.

Cell lysates were collected and quantified as total protein content. Twenty to thirty micrograms of total protein for each sample were separated by 10% SDS-PAGE and transferred to Westran S membrane (Whatman Inc.), followed by incubation with primary and secondary antibodies. Primary antibodies were mouse anti-p53 and anti-E7 from Santa Cruz Biotech; rabbit anti-c-Met, rabbit anti-EGFR, and mouse anti-HER3, rabbit anti-Akt, rabbit anti-phospho Akt, rabbit anti-Erk, and rabbit anti-phospho Erk from Cell Signaling; and mouse anti-β-actin from Sigma Aldrich. Secondary antibodies were purchased from Santa Cruz Biotech.