Affinipure goat anti rabbit secondary antibody

Tissue samples from the distal ileum were homogenized in RIPA lysis buffer containing 1% protease inhibitor (Beyotime, China), centrifuged at 10000 g for 10 minutes at 4°C and heated at 100°C for 10 minutes. Protein (30 μg) was then loaded into a 10% SDS-PAGE gel for electrophoretic separation and transferred to a PVDF membrane. The membrane was blocked with 5% fat-free milk for 2 h and incubated with 1 : 1000 dilutions of antibodies against β-actin (Proteintech Group), claudin-1 (Abcam, USA), ZO-1 (Proteintech Group, USA), and occludin (Abcam, USA) overnight at 4°C. The next day, the membrane was washed 4 times with Tris-buffered saline with Tween-20 (TBST) buffer and incubated with peroxidase-conjugated AffiniPure Goat Anti-Rabbit secondary antibody (Jackson ImmunoResearch, USA) for 90 min at room temperature. Protein bands were visualized with chemiluminescent HRP Substrate (Immobilon, USA). Protein concentration was measured using a BCA kit (YEASEN, China).