Goat anti rabbit igg

Cells were fixed with 95% ethanol, washed with phosphate-buffered saline (PBS) three times, blocked in non-immune goat serum at 37 °C for 30 min, and incubated with primary Trx1 antibody (1: 200, Abcam, Cambridge, UK) at 4 °C overnight. After washing thrice with PBS, the sections were incubated with fluorescein isothiocyanate (FITC) -conjugated secondary antibodies, goat anti-rabbit IgG (Cowin Biotech Co., Ltd., Beijing, China), at a 1: 25 dilution at 37 °C for 1 h. Cell images were obtained using an inverted fluorescence microscope (Carl Zeiss, Axiovert 200, Oberkochen, Germany), and analyzed using Image-Pro Plus 6.0 (Media Cybernetics, Inc., Rockville, MD, USA).

Proteins extracted from the colon tissue were separated by 8–12% SDS-PAGE after the concentration was detected by the BCA protein assay kit (CW0014S, CoWin Biotech Co., Inc., Beijing, China). They were then transferred to 0.2 μm polyvinylidene fluoride membranes (Merck KGaA Co., Ltd., Darmstadt, Germany). Membranes were blocked with 5% skim milk for 1.5 h. After blocking, the membranes were incubated with different primary antibodies at 4 ℃ overnight. Claudin 1 (1/3000, Abcam Co., Inc., Cambridge, UK), Claudin 3 (1/1000, Abcam Co., Inc., Cambridge, UK), Occludin (1/1000, Abcam Co., Inc., Cambridge, UK), ZO-1 (1/1000, Abcam Co., Inc., Cambridge, UK), TRL-4(1/1000, Proteintech Co., Wuhan, China), IKB(1/1000, Abmart Co., Inc, Shanghai, China), p65(1/5000, Abmart Co., Inc, Shanghai, China), p-IKB(1/1000, Abcam Co., Inc., Cambridge, UK), pp65(1/1000Abcam Co., Inc., Cambridge, UK), β-actin(1/1000, LABLEAD Biotech Co., Ltd., Beijing, China). Then, the membranes were washed with Trisbuffered saline Tween (TBST) and then incubated with horseradish peroxidase-conjugated goat anti-mouse IgG, or goat anti-rabbit IgG (CoWin Biotech Co., Inc., Beijing, China) for 1.5 h. The membranes were imaged with a Tanon 5200 imaging system. (Tanon Science & Technology Co., Ltd., Shanghai, China)