Nbp1 89702

Tumor tissues were dissected, embedded in OCT compound (4583, Sakura), and frozen immediately at −80°C. Serial 8-μm sections were collected from the cryostat. After fixation in 4% paraformaldehyde for 20 min and blocking in 10% donkey serum in 1× PBS at room temperature for 1 hour, the sections were permeabilized in PBS containing 0.1% Triton X-100. The sections were then incubated overnight at 4°C with the following primary antibodies: anti–E-cad (1:200; 610181, BD Biosciences), anti-WNT5A (1:50; ab235966, Abcam), anti-NRG1 (1:100; ab191139, Abcam), anti–α-SMA (1:200; A5228, Sigma-Aldrich), anti-ERBB3 (1:100; 12708, Cell Signaling Technology), anti-PDGFRα (1:200; 3174, Cell Signaling Technology), and anti-FZD6 (1:100; NBP1-89702, Novus Biologicals). Tissue sections were washed three times with PBS and incubated with the appropriate Alexa Fluor–conjugated secondary antibodies [donkey anti-rabbit Alexa Fluor 488 (1:500; A-21206, Invitrogen), donkey anti-mouse Alexa Fluor 568 (1:500; A10037, Invitrogen), donkey anti-mouse Alexa Fluor 568 (1:500; A10042, Invitrogen), or donkey anti-mouse Alexa Fluor 488 (1:500; A-21202, Invitrogen)] for 1 hour at room temperature, after which they were incubated with DAPI for 15 min. Images were obtained on a TissueGnostics imaging system (TissueFAXS Plus, Austria).

Tumor tissues were embedded in paraffin, sectioned, deparaffinized in xylene, and rehydrated in graded alcohol to distilled water. Slides were heated for antigen retrieval as follows: sodium citrate buffer (pH 6) for WNT5A, NRG1, PDGFRα, α-SMA, and FZD6 and tris-EDTA buffer (pH 9) for ERBB3. The endogenous peroxidase activity was blocked by incubation with 3% hydrogen peroxide for 10 min. The sections were incubated overnight at 4°C with the following primary antibodies: anti-WNT5A (1:100; ab235966, Abcam), anti-NRG1 (1:100; ab191139, Abcam), anti–α-SMA (1:5000; A5228, Sigma-Aldrich), anti-ERBB3 (1:50; 12708, Cell Signaling Technology), anti-PDGFRα (1:100; 3174, Cell Signaling Technology), and anti-FZD6 (1:100; NBP1-89702, Novus Biologicals). Tissue sections were washed three times with PBS, incubated with the appropriate horseradish peroxidase–conjugated secondary antibodies for 1 hour at room temperature, and detected using the 3,3'-diaminobenzidine (DAB) Substrate Kit (SK-4100, Vector). The sections were dehydrated and mounted with mounting medium (10004160, Sinaharm).