At 8 days after the S. enterica and E. hirae infections, intestinal tissues were fixed in 10% (w/v) PBS-buffered formaldehyde and 7 μm sections were prepared from paraffin embedded tissues. Immunohistochemical localization was performed as previously described [42 (link)]. Sections were incubated overnight with (1) purified goat polyclonal antibody directed towards ZO-1 (Santa Cruz Biotechnology, Santa Cruz, CA, USA, 1:500 in PBS, v/v) or (2) with purified hamster anti-Occludin (Santa Cruz Biotechnology, 1:500 in PBS, w/v). Sections were washed with PBS and incubated with secondary antibody. A biotin-conjugated goat anti-rabbit IgG and avidin–biotin peroxidase complex (Vector Laboratories, Burlingame, CA, USA) detected the specific labeling. The software Optilab Graftek (Graftek, Mirmande, France) assessed densitometry of immunohistochemistry photographs.
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