Quantitative Histological Analysis of Mucosal Immune Markers

Digitally fixed images of mucosal tunica were analyzed at 200× and 1000× magnification using an optical microscope (Olympus, Hamburg, Germany) equipped with an image analyzer (Image Pro Premier 9.1, MediaCybernetics, Rockville, MD, USA). The immunohistochemical data for TLR4, NF-kB p65, NF-kB p50, COX2, iNOS, TNF-α, IL-1β, Cas-3 and Cas-8 were qualitatively evaluated by a blinder examiner and were expressed as negative (−), very weak (−/+), weak (+), moderate (++) and strong (+++) positivity. Additionally, a blinder examiner evaluated immunostaining also quantitatively, as Integrated Optical Density (IOD) using an image analyzer (Image Pro Premier 9.1, MediaCybernetics, Rockville, MD, USA) [47 (link),48 (link)]. The data were pooled to represent a mean value and a statistical analysis was applied to compare the results obtained from the different experimental groups. The analysis was performed on five sections for each sample spacing 70 μm and evaluating six random fields with the same area (52 × 10³ μm²) per section.