Ab216462

Tissue sections were deparaffinized, rehydrated, and subjected to antigen retrieval, then stained with primary antibodies as flows: neutrophil gelatinase-associated lipocalin (NGAL, 1:200, Ab216462, Abcam, UK), 4-HNE (1:200, MAB3249-SP, NOVUS, USA), according to the IHC kit instructions (DAB-0031, KIT-9710, MXB, China). Samples were dehydrated, cleared, and sealed and representative images captured by microscopy (DP73, OLYMPUS, Japan; magnification, 400×). NGAL and 4-HNE IOD values were calculated as for Prussian blue staining.

The mRNA expression levels of LCN2 in the normal, acute colitis, chronic colitis, and CAC mice were detected by RT-PCR. The LCN2 primer used for the amplification was as follows: 5′- CACCACGGACTACAACCA-3′ and 5′-ACACTCACCACCCATTCA-3′ (Sangon Biotech, China). Here, β-actin was regarded as the internal control. The reliability of the PCR results was verified in correspondence with the dissolution curve. The cycle threshold (Ct, which is the inflection point on the amplification power curve) was calculated, and the relative gene expression was calculated using the 2^-ΔΔCt method [ΔCt = Ct (target gene) - Ct β-actin, ΔΔCt = ΔCt (target gene) - ΔCt (β-actin)]. Five samples of each group were randomly chosen for RT-PCR. The protein expression levels of LCN2 in the normal, acute colitis, chronic colitis, and CAC mice were detected by Western blot. The Anti-LCN2 antibody (ab216462, 1:1,000) and the Anti-GAPDH antibody (ab9485, 1:2,500) were purchased from Abcam. The target protein bands were developed by using enhanced chemiluminescence Western blotting detection reagent, and densities were quantified using Total Lab Quant V11.5 (Newcastle upon Tyne, UK). Three samples of each group were randomly chosen for Western blotting analysis.