Anti ki67

Immunohistochemistry was performed as reported previously (Hoare et al., 2016 (link)). Formalin fixed paraffin-embedded mouse tissues were stained with the following antibodies: anti-Cox2 (as above); anti-NRAS (Santa Cruz, sc-31,1:100); anti-p21 (BD, 556431, 1:50); anti-ki67 (Bethyl, IHC-00375, 1:1000); anti-Ly6C (Abcam, ab15627, 1:400); anti-Cd11c (Cell Signaling, 97585, 1:350); anti-Cxcl1 (Abcam, ab86436, 1:100); anti-PGE₂ (Abcam, ab2318, 1:100); anti-Foxp3 (eBioscience, 14-5773, 1:100); after proteinase K digestion (Ly6C) or heat-induced epitope retrieval in citrate (pH6) or Tris-EDTA (pH9) buffers before visualization using the DAKO Envision kit according to manufacturer’s instructions and counterstaining with hematoxylin. For fluorescent labeling we utilized appropriate fluorochrome-tagged secondary antibodies (Life Technologies). For EdU staining (ThermoFisher C10638), the same protocol was followed, with the following extra step: after antigen retrieval, 3% BSA washes in PBS were performed twice, and CliCK-iT reaction cocktail was added for 30 min following the manufacturer’s instructions.
All slides were scanned on a Leica AT2 at 20x magnification and a resolution of 0.5 μm/pixel. Following digitization, image analysis was performed as described previously using HALO (Indicalabs) (Hoare et al., 2016 (link)).

Chemicals and reagents were obtained from the following sources: Matrigel from Corning (#356,231), azoxymethane (AOM) from MRIGlobal (#0061); and dextran sodium sulfate (DSS) from MP Biomedical (#160,110). If not otherwise specified, chemicals and reagents were from Sigma-Aldrich, St. Louis, MO, USA. Antibodies were obtained and used as follows: anti-βPix antibodies from Millipore (#07–1450-I; used for immunoblotting), Santa Cruz (#sc-393184; used for immunohistochemistry in murine tissue), and Sigma-Aldrich (#HPA004744; used for immunohistochemistry in human tissue); anti-total β-catenin from Abcam (#ab19381); anti-activated-β-catenin from Millipore (05–665-AF555); Alexa Fluor 488 from Invitrogen (#A21206); Alexa Fluor 594 from Invitrogen (#A21203); anti-GAPDH from Cell Signaling (#2118); anti-FLAG antibody from Sigma-Aldrich (#F1365); and anti-Ki67 from Bethyl Labs (#IHC-00375). PCR primers are shown in Supplemental Table 1.

The collected organs were formalin-fixed and paraffin-embedded (FFPE) for routine histology. To assess the percentage of PNLs, the entire cerebellum was examined, recovering FFPE brain sections with intervals of 70 μm. At the recognition of abnormal cerebellar regions—in general, defined as PNLs—sections were collected and stained with hematoxylin and eosin (H&E). PNLs cross-sectional areas were carried out using imaging software NIS-Elements BR 4.00.05 (Nikon Instruments Europe B.V., Campi Bisenzio, Italy).
For immunohistochemistry, PNL-positive FFPE brain sections (4 μm) were dewaxed for 20 min at 56 °C and incubated in citrate buffer pH 6.0 for 20 min at 95 °C. After peroxidases inhibition by 3% H₂O₂ for 10 min, sections were incubated with primary antibodies anti-Ki67 (Bethyl, Montgomery, TX, USA), anti-NeuN (Merck Millipore, Darmstadt, Germany), and anti-caspase-3-activated (Cell Signaling Technology, Danvers, MA, USA) for 1 h at room temperature in a moist chamber. After incubation with the secondary anti-rabbit antibody (Bethyl), the antigen–antibody reaction was revealed by DAB (Dako, Agilent Technologies, Santa Clara, CA, USA) and analyzed by HistoQuest (TissueGnostics, Vienna, Austria) software.