2.12. Western Blot Analysis

Organoids were analysed for TJ expression by Western blot analysis after treatment with LPS or EcN. Organoids were homogenized with a tissue homogenisator (Ultra Turrax, IKA®-Werke GmbH & Co. KG). Proteins were extracted and separated by sodium dodecyl sulfate polyacrylamide gel electrophoresis, transferred to a nitrocellulose membrane, and immunoblotted using primary antibodies against occludin, claudin8, zonula occludens-1 (Invitrogen), and claudin4 (Abcam). Immunoblotting for GAPDH (GenScript USA Inc., NJ, USA) was used as an internal control. After incubation with the secondary antibody (donkey anti-rabbit IgG (HRP); Abcam), the membrane was developed with a chemiluminescence solution (Bio-Rad Laboratories, Hercules, California, USA) and images were acquired using a ChemiDoc™ Touch Imaging System (Bio-Rad). Blot analysis was performed via ImageJ software (open source).