Envision system horse radish peroxidase labelled polymer

Immunohistochemical staining was performed using the Dako autostainer platform (Dako, Denmark) as previously described [24 (link)]. Sections were stained using the following primary antibodies: mouse anti-CD68 (clone PG-M1, 1:100, Dako), mouse anti-CD45 (clone 2B11, 1:200, Dako), rabbit anti-Iba1 (ionized calcium binding adaptor molecule 1, 1:1000, Wako), rabbit anti-GFAP (1:2000, Dako), mouse anti-neurofilament (NF) (phosphorylated and non-phosphorylated NF-heavy chain; clone N52, 1:1000, Sigma-Aldrich), mouse anti-IL-1α (clone 4414, 1:1200, R&D Systems), mouse anti-IL-1β (clone 2E8, 1:50, BioRad), rabbit anti-TNF (1:100, ThermoFisher Scientific), rabbit anti-TNFR1 (clone H-271, 1:50, Santa Cruz), rabbit anti-TNFR2 (1:50, Sigma-Aldrich), and rat anti-IL-1Ra (clone 40,007, 1:1500, R&D Systems). The antigen-antibody complex was visualized using EnVision+System horse-radish peroxidase-labelled Polymer (Dako), PowerVision+Poly-HRP IHC (AH Diagnostics), or CSAII (Dako) detection systems.

The experiments on paraffin-embedded colonic tissues and TMAs were approved by umbrella spore IRB no. 070166. Tumour xenografts were fixed in neutralized formalin and embedded in paraffin. Slices were deparaffinized with serial histoclear and ethanol. Antigen retrieval was performed in citrate buffer (pH 6.0) with high pressure at 110 °C for 15 min and then quenched in 0.03% H₂0₂ with sodium azide for 5 min. The slides were incubated with primary antibodies at room temperature for 60 min and then incubated in Dako Envision + system horseradish peroxidase-labelled polymer at room temperature for 30 min. Signal was detected by incubating in a DAB+ substrate chromogen system at room temperature for 5 min. The primary antibodies used were: anti-DPEP1 (1:1,000; Sigma-Aldrich, HPA012783), anti-CD73 (clone D7F9A, 1:300; Cell Signaling Technology, 13160), anti-TGFBI (clone EPR12078(B), 1:300; Abcam, ab170874), anti-FASN (clone G-11, 1:500; Santa Cruz Biotechnology, sc-48357) and anti-AGO2 (clone EPR10411, 1:500; Abcam, ab57113).