Vegf a

ELISA kits for MPO, VEGF-A, TGF-β, IL-4, IL-1β, IL-6, TNF-α, and IFN-γ were purchased from CUSABIO company. According to the manufacturer's instructions, colon tissues were broken into small pieces and homogenized in PBS solution using a tissue homogenizer. After centrifugation at 5000 x g for 5 min at 4 °C, the supernatants were collected and stored at -20 °C or -80 °C.100 μl of standard and sample were added to each well and incubated for 2 h at 37 °C. After removing the liquid, 100 μl of Biotin-antibody (1x) was added to each well and incubated for 1 h at 37 °C. After washing, 100 μl of HRP-avidin (1x) was added to each well and incubated for 1 h at 37 °C. 90 μl of TMB Substrate was added to each well and incubated for 15 min at 37 °C in the dark. Then, 50 μl of Stop Solution was added to each well and incubated for 5 min. OD values were measured using a microplate reader at a wavelength of 450 nm, and standard curves were established to measure the concentrations of MPO, VEGF-A, TGF-β, IL-1β, IL-6, and TNF-α in each sample.