Anti pge2

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)).

Protein samples isolated from bladder tissues and cells were separated by 10% SDS-PAGE and transferred onto nitrocellulose membrane. The membranes were blocked in 5% bovine serum albumin and probed with specific antibodies: anti-TPRG1 and anti-COX-2 (1:1500, Abcam), anti-TNF-α and anti-IL-6 (1:2000, Abcam), anti-IL-1β and anti-β-actin (1:2500, Abcam), anti-p65 and anti-PGE2 (1:3000, Abcam). A Cell Nuclear and Cytoplasmic Protein Extraction Kit (Beyotime) was used to isolate nuclear and cytoplasmic proteins from the pCGs, and the proteins were also separated and transferred. The membranes were also probed with specific antibodies: anti-p65 and anti-β-tubulin (1:3500, Abcam), anti-Histone H3 (1:4000, Abcam). Following incubation with horseradish peroxidase-conjugated secondary antibody (1:4500, Abcam) and tetramethylbenzidine, the immunoreactivities were visualized using enhanced chemiluminescence (Sigma-Aldrich).