Alexa fluor 594 goat anti mouse or rabbit igg

Epithelial cell layers were stained using the following primary antibodies: ZO-1 (1:100; BD Biosciences, NJ, USA). The secondary antibodies used were Alexa Fluor 488 goat anti-mouse or rabbit IgG, and Alexa Fluor 594 goat anti-mouse or rabbit IgG (1:500; Thermo Fisher Scientific, MA, USA). To determine occludin expression and cellular distribution, an occludin mouse monoclonal antibody (OC-3F10) was used as an Alexa Fluor® 594 Conjugate (Thermofischer). Nuclei were stained using DAPI (4′,6-Diamidin-2-phenylindol, conc. 1:2000). Immunofluorescence staining was analyzed by confocal laser scanning microscopy (LSM 780, Carl Zeiss, Jena, Germany) as previously described [9 (link),11 (link)].

T84 monolayers were washed with PBS and fixed with 1% paraformaldehyde. Paraffin-embedded mouse tissues were derived from experiments performed by Schneditz and colleagues. In short, antibiotic-induced dysbiosis was simulated in C57BL/6 mice by amoxicillin/clavulanate and indometacin treatment and intragastrical infection with AHC6 or Mut-89, leading to K. oxytoca overgrowth in the colon of infected mice [8 (link)]. Caecum sections were cut, deparaffinized, and heated in sodium citrate buffer solutions at pH 6.0 for epitope retrieval. After permeabilization with 0.05% Triton X-100, immunostaining of TJ proteins was carried out using anti-ZO-1 or anti-occludin, anti-claudin-1, -5, or -8 (1:100), followed by labelled Alexa Fluor 488 goat anti-mouse or rabbit IgG and Alexa Fluor 594 goat anti-mouse or rabbit IgG (1:1000; Thermo Fisher Scientific). Staining of ZO-1 and claudin-1 on mouse tissue sections was limited to high background staining and weak antibody binding. Therefore, occludin was used in this case. Nuclei were DAPI stained (1:5000). Fluorescence staining was visualized by confocal laser scanning microscopy (LSM 780, Zeiss, Jena, Germany), and tight junction proteins were localized by z-stack imaging. Distribution profiles of claudin signals were generated using Zen software (ZEN 2.3 lite, Zeiss, Oberkochen, Germany).