Epr1344

Paraffin-embedded colon sections were dewaxed, rehydrated, and pre-treated with hydrogen peroxidase in PBS buffer. Heat-induced antigen retrieval was performed. After blocking with the appropriate antisera in blocking buffer, sections were incubated with anti-Proliferating cell nuclear antigen (PCNA) (Thermo scientific, clone PC10, 1:300 dilution), anti-β-catenin (Cell Signaling Technology, CST, clone 6B3, 1:100 dilution), anti-p53 (Leica-microsystems, clone CM5, 1:100 dilution), or anti-COX-2 (Thermo scientific, 1:100 dilution), anti-E-cadherin (CST, clone 24E10, 1:200 dilution), anti-N-cadherin (Millipore, clone EPR1792Y, 1:50 dilution), anti-Fibronectin (Epitomics, clone F14, 1:200 dilution) and anti-Vimentin (CST, clone D21H3, 1:50 dilution). After incubation with HRP-conjugated secondary antibody and tyramide amplification followed by streptavidin-HRP, positive signals were visualized by DAB kit , and counter-stained with hematoxylin. Double staining of CD11b⁺Ly6g⁺ cells was carried out using Polink RRt DouSP kit (ZSGB-BIO ORIGENE, China). CD11b (abcam, clone EPR1344, 1:100 dilution), Ly6g (abcam, clone RB6-8C5, 1:20 dilution). Five randomly selected fields from each section were examined at a magnification of 400× and analyzed using NIS-Elements. The positive content was calculated using the following formula: positive content (PC) = mean optical density × positive area.

Cancer tissues were obtained from colorectal surgical specimens (n = 7). The tumors of all colorectal cancer patients were low grade (well or moderately differentiated). Colon specimens were fixed in formalin and embedded in paraffin. After deparaffinization, antigen retrieval was conducted at 95 °C for 20–30 min in EDTA buffer (pH 9.0). Endogenous peroxidase activity was blocked with 0.3% H₂O₂ at room temperature for 10 min. After protein blocking at room temperature for 10 min, slides were incubated with a polyclonal rabbit anti-human IL-11 (RPA057Hu01, Uscn Life Science, USA), a polyclonal rabbit anti-human phospho-STAT3 (Tyr705) antibody (9145, Cell Signaling Technology), a monoclonal mouse anit-CD14 antibody (MY4), or a rabbit monoclonal anti-CD11b antibody (EPR1344, Abcam, Cambridge, UK) overnight at 4 °C. Sections were then incubated at room temperature for 15 min with a horseradish peroxidase-labelled anti-rabbit Igs. Positive signals were amplified with the CSA II Biotin-free Tyramide Signal Amplification System (Dako, Japan) and visualized using 3-3′-diaminobezidine-4HCL (DAB). Hematoxylin-eosin staining was then performed according to standard procedures.