Hm550 vpd

Renal tissues from mice were rapidly frozen and mounted in Optimal Cutting Temperature compound (Sakura Finetek Japan, Co., Ltd., Tokyo, Japan). Sections (4 μm thick) were prepared using a cryostat (HM550-VPD; Thermo Fisher Scientific) and mounted onto glass slides. SA-β-gal activity was measured using a senescence detection kit (BioVision Inc., Milpitas, CA, USA) according to the manufacturer’s protocols. Samples were viewed under bright field at ×100 magnification using the BZ-9000 microscope (Keyence Corp.).

Tumours arising from Cle-H3 cells with or without the expression of RNF43(3SA) mutant were fixed with 10% formalin and subsequently sectioned at 7 μm using a cryostat (HM550-VPD, Thermo Fisher Scientific). Sections were treated with 0.1% Triton-X in PBS for permeabilization and then with LAB solution (Polyscience, #24310) for antigen retrieval. Each sample was stained with anti-β-catenin mouse IgG1 (BD-TDL #199220) at 1:500 dilution, anti-Vimentin mouse IgM (Sigma, #V5255) at 1:2000 dilution and DAPI (1 μM) in combination with anti-mouse IgG1-Alexa555 (Invitrogen, #A-21127) at 1:1000 dilution and anti-mouse IgM-Alexa488 (Invitrogen, #A-21042) at 1:1000 dilution in 1.5% NGS and 0.1% BSA in TBST after blocking with 5% NGS in TBST. Tumour images were taken using a confocal laser scanning microscope (Carl Zeiss, Axio Imager Z1 & LSM700) equipped with a water-immersion ×40 objective lens (C-Apochromat 40 × /1.20 W Corr M27) and ZEN Black 2011 software (Zeiss). Z-stack images were processed and arranged using ImageJ software (1.52 v, NIH) and Photoshop CS5 (12.0 × 64, Adobe).