Immunohistochemical Analysis of Tight Junction Proteins

The colon tissues were fixed in 4% paraformaldehyde, embedded in paraffin, dewaxed in xylene and rehydrated in gradient alcohols. The tissue sections were retrieved in citrate buffer (pH 6.0) and incubated with primary antibodies (rabbit anti-occludin, 1:500, GB111401, Servicebio; rabbit anti- zonula occludens-1, 1:200, A0659, Abclonal; rabbit anti-claudin, 1:500, GB11032, Servicebio) overnight at 4 °C. Next, these sections were incubated with horseradish peroxidase (HRP)-conjugated goat anti-rabbit IgG (1:200, GB23303, Servicebio, China) at 25 °C for 2 h. After washing in PBS, the sections were counterstained with haematoxylin for 3 min, dehydrated and mounted. For quantification, five fields were randomly selected for each sample. The expression of occludin, ZO-1 or claudin protein was quantified by the histochemistry score (H-score) by selecting five regions on each slice. The average H-score values from the five regions were statistically analysed to determine the overall percentage of occludin, ZO-1 or claudin protein-positive area across the four groups. H-score = ∑ (pi × i) = (percentage of weak intensity × 1) + (percentage of moderate intensity × 2) + (percentage of strong intensity × 3)^{54 (link),55 (link)}.