Anti mouse igm alexa488

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).

For FACS analysis, the following fluorochrome-labeled antibodies specific for mouse molecules were used: CD4-FITC, CD4-Alexa647, CD19-Pacific blue (BioLegend, Inc., San Diego, CA, USA), and GL7-FITC and FAS-PE (Becton Dickinson Ltd., Franklin Lakes, NJ, USA). For immunohistochemistry, anti-mouse-IgM-Alexa488, anti-rat-IgG-Alexa488, and anti-mouse-IgG-Alexa555 (Invitrogen Life Technologies, Carlsbad, CA, USA), anti-mouse-IgD-biotin (BioLegend), GL7-Alexa488 (eBiosciences Inc., San Diego, CA, USA), and anti-mouse IgM F(ab’)₂ Alexa647 (Jackson ImmunoResearch laboratories Inc., West grove, PA, USA) were used.