Anti st2 antibody

Immunohistochemistry staining was performed as described previously [31 (link)]. The tumors removed from the wild-type or IL-33 transgenic mice at Day 22 post tumor inoculation were fixed with 4% formaldehyde and embedded with paraffin. The sections were labeled with anti-Ki67 antibody (Arigo, 1:200) and anti-PCNA antibody (Boster, 1:200). Quantification of Ki67 and PCNA expression was independently performed by two pathologists. The positively staining cells were quantified by ImageJ software. Twenty CRC tissues and adjacent normal tissues were obtained from surgery in Union hospital (Wuhan) with the permission of each patient. The ST2 staining was performed with anti-ST2 antibody (R&D, 1:200).

Bone marrow (BM) cells from the femur and tibia were isolated and red blood cells were lysed. BM cells were cultured in DMEM-F12 media supplemented with 10%FBS and 20% supernatant derived from CMG14–12 (M-CSF producing) cell line for 5–7 days prior to experiments. BMDM polarization was assessed by cell morphology and later confirmed by cell surface expression of CD64, F4/80, CD11b, and CD11c. For some experiments, BMDMs were exposed to neuron-derived supernatants overnight. On the day of experiment, cells were exposed to vehicle, 50 or 250ng/mL of LPS (Sigma-Aldrich) for 6–8hours. BD GolgiPlug™ Protein Transport Inhibitor (containing Brefeldin A) and BD GolgiStop^™ Protein Transport Inhibitor (containing Monensin) were added in the last 5 hours of stimulation. Cells and supernatant were recovered to be analyzed for cytokine expression by flow cytometry and cytokine concentration by ELISA, respectively. For IL-1β release, BMDMs were treated with 250ng/mL of LPS (Sigma-Aldrich) for 4 hours followed by exposure to vehicle, 0.5 or 2.5mM of ATP(Sigma-Aldrich). Supernatants were assessed for cytokine concentration by ELISA. For some experiments, control BMDMs were treated with 500ng/mL of neutralizing anti-ST2 antibody (R&D systems) or CD11c-IL-33KO BMDMs were exposed to 500ng/mL of mouse rIL-33 (R&D systems) 24hours prior to LPS stimulation.