β catenin antibody

Immunocytochemistry for CC3 and β‐catenin was performed to analyse cell death after 24‐hr stimulation with Wnt ± H₂O₂ or to analyse β‐catenin localization after 30 min. VSMCs were fixed with 3% paraformaldehyde/PBS and permeabilized with 0.1%–0.2% Triton X‐100/PBS. After blocking with 20% goat serum/PBS, 1 µg/ml CC3 antibody (AF835; R&D Systems) in 1% BSA/PBS or 2.5 µg/ml β‐catenin antibody (610154; BD Transduction Laboratories, Oxford, UK) in PBS was added overnight at 4°C. Bound antibodies were detected with biotinylated goat anti‐rabbit IgG (B7389; Sigma‐Aldrich) or biotinylated goat anti‐mouse IgG (BA9200; Vector Laboratories, Peterborough, UK) and then DyLight‐488 Streptavidin (SA‐5488‐1; Vector Laboratories) diluted 1:200 in PBS. Coverslips were mounted in ProLong Gold and DAPI (P36931; Invitrogen, Paisley, UK).

Histological assessment was performed following standard hematoxylin and eosin (H + E) staining and in conjunction with a dermatopathologist (A.H.). Immunofluorescent labeling with Antibodies against DNMT3A, β-catenin, and Ki-67 was performed^{7 (link)}. Tissue sections from snap frozen skin tumor biopsies were fixed, blocked, and then probed overnight at 4 °C with primary antibodies. Antibodies against DNMT3A (#3598) and Ki-67 (#9449) were obtained from Cell Signalling, USA. β-Catenin antibody (#610153) was obtained from BD Transduction USA. Secondary fluorescent antibodies (Alexa Fluor #111-5451144 488-conjugated goat-anti-rabbit and #115-585-146 594-conjugated goat-anti-mouse) were applied the following day and visualized with a fluorescent microscope (Zeiss Axioimager Z2, with Apotome 2—Carl Zeiss, UK).

SW480 and SW620 cells (2 × 10⁴) were seeded on 12 mm coverslips until 60% confluence and then transfected as described previously. Non-transfected cells were used as an additional control (only kept in Opti-MEM™ medium during transfection). After transfection, cells were fixed in 4% paraformaldehyde for 30 min. Fixed cells were then permeabilized with 0.1% Triton X-100 in PBS for 10 min, washed, blocked with 3% BSA in PBS for 30 min, and incubated for 15 h with β-catenin antibody (1:100, BD transduction 610154 Mouse antibody). Cells were afterward washed in PBS and incubated with Alexa Fluor 488 secondary antibody (Goat anti-Mouse IgG. 1:200, Cat.#. A21207, Life Technologies) for 1 h. The coverslips were counterstained with 4′,6-diamidino-2-phenylindole (DAPI) and imaged under a confocal microscope C2+ (Nikon, Japan). Images were processed and analyzed using the software ImageJ Ver.2.3.0/1.53q (NIH, Bethesda, MD, USA.). Colocalization images were obtained using the Colocalization Threshold tool in ImageJ.

Formalin fixed and paraffin embedded slides were first treated in antigen retrieval buffer at 98C for 15 minutes, and then incubated with primary antibody at 4C overnight or 1 hour at room temperature. The next day, after washing, the samples were incubated with HRP conjugated secondary antibody for 30 minutes. They were then washed and incubated with DAB under close monitoring for color development. The β-Catenin antibody was purchased from BD Transduction Laboratories (Cat# 610153), the Ki-67 antibody was purchased from Biocare Medical (Cat# CRM325), the Cyclin D1 antibody was purchased from Millipore (Cat# 04-1151), the Cyto-keratin antibody was purchased from Dako Cytomation (Cat# Z0622), the HGF antibody was purchased from R&D Systems (Cat# AF-294-NA), and the phosphor-Met antibody was purchased from Cell Signaling (Cat# 3077). Mucicarmine staining kit was purchased from Dako Cytomation (Cat# SL016).