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The sections were then incubated with primary antibodies against occludin (Abcam, dilution 1:100) and ZO-1 (ThermoFisher, dilution 1:100), diluted in PBS containing 0.2% BSA and 0.1% saponin at 4 C overnight, washed, and incubated for 1 h with Alexa Fluor 647 donkey anti-rabbit IgG secondary antibody (Invitrogen, dilution 1:400)