Fluorochrome tagged secondary antibodies

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)).