Ab256524

Immunohistochemistry was performed as previously described with DAB2 rabbit monoclonal antibody (1/800, ab256524, Abcam, Cambridge, UK) [37 (link)]. Slides were scanned by NanoZoomer Digital Pathology System (Hamamatsu Photonics, Hamamatsu City, Japan). DAB2 immunostaining H-score in 3 tumor and stroma areas per core was quantitated using QuPath software (version 0.2.3) and maximum H-score was assessed in relation to patient prognosis and relapse [2 (link)]. For immunofluorescence, tissue sections from matching primary and metastatic cancers (n = 5) were incubated overnight at 4 °C with DAB2 (1/100, ab256524, Abcam) or CD68 mouse monoclonal antibody (1/400, ab955, Abcam). Positive cells were detected with α-rabbit-IgG (H + L) AlexaFluor™ Plus 594 (1/400, A23740, Invitrogen) or α-mouse-IgG (H + L) AlexaFluor™ Plus 488 (1/400, A23723, Invitrogen). Nuclei were visualized with DAPI (1.5 µg/mL, Molecular Probes, Life Technologies) as previously described [31 (link)]. Tissues were imaged with the BX50 epifluorescence microscope (40X objective, Olympus, Tokyo, Japan). Number of DAB2 positive ( +), CD68 + and double positive (DAB2 + CD68 +) cells in both tumor epithelium and tumor associated stroma areas were counted by two individual assessors in 3–8 sections/tissue. Number of positive cells was normalized to area of tumor epithelium or stroma (µm²).

ES-2 and ES-2:ES-2-Rv-NICD3 cells were plated at 2 × 10⁵ cells/well in 6 well plates and cultured 48 h before treatment with the hyaluronan synthesis inhibitor 4-methylumbelliferone (4-MU, 1 mM, Sigma-Aldrich) or vehicle control (PBS) for 24 h [37 (link)]. Protein lysates from monolayer and spheroids were prepared in RIPA buffer as described previously [59 (link)]. ES-2, A2780 and OVCAR3 cells were cultured to 80% confluency before protein isolation. 20 µg of protein was electrophoresed on 4–20% TGX gels (Bio-Rad, Hercules, CA, USA) and transferred overnight to PVDF membrane (GE healthcare, little Chalfont, England). Proteins were detected with DAB2 rabbit monoclonal antibody (1/2000, ab256524, Abcam), anti-rabbit IgG peroxidase conjugated antibody (1/4000, Sigma-Aldrich) and Amersham™ ECL™ Prime (Cytvia, Marlborough, MA, USA). Chemiluminescence was detected using the ChemiDoc™ Imaging System (Bio-Rad) and band intensity was calculated with ImageLab software (Bio-rad, version 6.1). β-actin monoclonal mouse antibody (1/5000, ab8226, Abcam) or GAPDH monoclonal mouse antibody (1/50,000, 60004-1-Ig, Proteintech®, Rosemont, IL, USA) was used as a loading control.