Ab72583

Immunohistochemical staining has been performed by using rabbit anti-human Wnt5a antibody (1:100, ab72583, Abcam, USA) and rabbit anti-human Ror2 antibody (1:200, HPA021868, Sigma, Germany). The sections were heated in tissue-drying oven for 1 h at 60 °C, routine deparaffinized, and rehydrated. Antigen retrieval was performed in a microwave for 30 min with citrate buffer, and it was cooled to room temperature and washed with phosphate-buffered saline (PBS). Endogenous peroxidase activity was blocked with endogenous peroxidase blockers for 30 min, then the peroxidase was incubated in 5 % normal goat serum for 30 min. After overnight incubation at 4 °C with the primary antibody, sections were washed with PBS and subsequently incubated with a biotin-conjugated secondary antibody for 1 h at room temperature. The reactions became visible after immersion of diaminobenzidine tetrahydrochloride (DAB).

Immunohistochemistry was performed as previously described^{34 (link)}. The primary antibodies used in the study included a rabbit monoclonal anti-β-catenin antibody (1:400; #8814; Cell Signaling), a rabbit polyclonal anti-Dickkopf-1 antibody (1:100; sc-25516; Santa Cruz), a mouse monoclonal anti-WNT16 antibody (1:100; sc-271897; Santa Cruz), and a rabbit polyclonal anti-WNT5a antibody (1:100; ab72583; Abcam). The biotinylated goat anti-mouse and goat anti-rabbit IgG were acquired from Boster (Wuhan, China). To obtain the percentage of cells that expressed a given marker protein, photomicrographic images of each section were captured with an Olympus microscope and a digital camera under ×200magnification. The number of specific antigen-positive cells was counted in five random fields. The mean and standard deviation of the percentage of positive cells were calculated for each group and were used for the statistical analysis.

Paraffin‐embedded tissue was section into 4‐μm‐thick sections, and stained. Briefly, sections were rehydrated and equilibrated in TBS before antigen retrieval, either by heat in low pH buffer, or enzymatically with Proteinase K (20 μg/ml, 37°C/30 min), before primary antibody was applied (omitted for controls) and incubated 1.5 h (RT). Slides were rinsed and secondary antibody, visualized using DAB, counterstained in HTX and mounted. The following antibodies were used to visualize markers of Wnt signaling: Wnt3a (1:300, 09‐162, EMD Millipore Corp. Billerica, MA), Wnt5a/b (1:100, ab72583, Abcam, Cambridge, UK), β‐catenin (1:100, AF1329, R&D Systems, Minneapolis, MN), Disheveled‐associated activator of morphogenesis 1/DAAM1 (1:200, ab71327, Abcam), Inhibitor of β‐catenin and TCF‐4/ICAT (1:50, ab197916, Abcam) and Nemo‐like kinase/NLK (1:100, ab26050, Abcam). For double stainings to visualize cell types, the following antibodies were used in combination with β‐catenin; Prosurfactant Protein C (ProSpC, 1:1000, ab90716, Abcam), pan‐cytokeratin (1:100, ab27988, Abcam) and Vimentin (1:500, ab92547, Abcam), as well as ICAT and NLK. Fluorescent secondary antibodies were used (dilutions and pretreatments were identical when applicable).