The colon specimens without fecal stream removed for histological analysis were
immersed in 10% neutral formalin buffer for 24 h and then dehydrated by exposure
to increasing ethanol concentrations, xylene, and embedded in paraffin.
Thereafter, the sections of tissue were cut at 5 μm and were mounted on a glass
slide, cleared, hydrated, and stained with H&E for the evaluation of the
presence of colitis and the degree of inflammation. The slides were analyzed
under an optical microscope (Eclipse F-50, Nikon Inc., Osaka, Japan) at a
magnification of 200×. The slides prepared for a pathologist who was unaware of
the objectives of the study evaluated both histology and immunohistochemistry
(anti-claudin-3 and anti-occludin).
Photomicrographs were taken with a digital video-capture camera (DS-Fi-50; Nikon
Inc., Osaka, Japan) coupled to the microscope body. The presence of colitis in
the colon segments devoid of the fecal stream was confirmed considering three
different histological parameters: mucosal-submucosal neutrophil infiltration,
presence of epithelial erosion and ulceration, and classified in crosses (- to
9+) for each variable. The severity of the inflammation in the colonic mucosa
devoid from the fecal stream was established in accordance with a previously
used inflammatory grade scale4 .
immersed in 10% neutral formalin buffer for 24 h and then dehydrated by exposure
to increasing ethanol concentrations, xylene, and embedded in paraffin.
Thereafter, the sections of tissue were cut at 5 μm and were mounted on a glass
slide, cleared, hydrated, and stained with H&E for the evaluation of the
presence of colitis and the degree of inflammation. The slides were analyzed
under an optical microscope (Eclipse F-50, Nikon Inc., Osaka, Japan) at a
magnification of 200×. The slides prepared for a pathologist who was unaware of
the objectives of the study evaluated both histology and immunohistochemistry
(anti-claudin-3 and anti-occludin).
Photomicrographs were taken with a digital video-capture camera (DS-Fi-50; Nikon
Inc., Osaka, Japan) coupled to the microscope body. The presence of colitis in
the colon segments devoid of the fecal stream was confirmed considering three
different histological parameters: mucosal-submucosal neutrophil infiltration,
presence of epithelial erosion and ulceration, and classified in crosses (- to
9+) for each variable. The severity of the inflammation in the colonic mucosa
devoid from the fecal stream was established in accordance with a previously
used inflammatory grade scale