Quantitative Claudin-1 Immunohistochemistry

Sections (epithelial cells) were incubated overnight at 4 °C with the primary anti-claudin-1 antibody. Sections were then exposed to appropriate biotinylated immunoglobulins, peroxidase-labelled streptavidin complex and 3.3′-diaminobenzidine tetrahydrochloride, counterstained and examined by a Leica DMRB light microscope equipped with a DFC480 digital camera (Leica Microsystems, Cambridge, UK). For each group, representative photomicrographs were shot and analysed quantitatively using the Image Analysis System “L.A.S. software version 4.5” (Leica Microsystems, Cambridge, UK). Two blind investigators (C.I. and C.S.) carried out cell-counting independently and assessed the colorimetric threshold values to detect antigen expression levels. Immunostaining expression was calculated as ratio between area of the stained fields and the total tissue area examined (percentage positive pixels (PPP)), as previously reported [58 (link),61 (link)]. Data obtained from all the examined fields for each rat were averaged and used to calculate mean values ± SEM for each experimental group, which were plotted in graphs.