Mouse anti cldn 2

After the exposure, organoids were rinsed with PBS, fixed with 4% (w/v) paraformaldehyde, permeabilized with 0.5% (v/v) Triton X-100 in PBS at RT for 30 min, and then blocked with 1% (w/v) BSA at RT for 1 h. Afterwards, the organoids were incubated at 4 °C for 36 h with mouse anti-CLDN-2 (1:100 dilution, Invitrogen) or overnight with mouse anti-E-cadherin (1:200 dilution, Abcam) following 1-h incubation of Alexa Fluor 488 goat anti-mouse IgG secondary antibody (1:200 dilution, Thermo Fisher Scientific). Organoids were then washed three times with PBS and stained with diamidino-2-phenylindole (DAPI, Sigma) at 1:1500 dilution. After another two washings with PBS, organoids were mounted using VectaShield mounting medium (Vector Laboratories, Burlingame, USA). Confocal images were obtained using a confocal microscopy (Leica Microsystems GmbH) with identical acquisition settings (laser power, objectives, magnifications) for each acquired image and condition. Images were then analyzed using the Image J software.

After the exposure, intestinal organoids were lysed in 100 μL RIPA buffer and the extracted proteins were denatured using SDS loading buffer (125 mM Tris–HCl pH 6.8, 4% sodium dodecyl sulfate, 20% glycerol, 0.04% bromophenol blue, and 100 mM β-mercaptoethanol) at 95 °C for 5 min. For each sample, 10 μg protein was loaded and separated on 10% or 12% mini-protein TGX precast protein gels (Bio-Rad) and transferred to polyvinylidene difluoride membranes (GE Healthcare, Chicago, USA). The membranes were incubated overnight at 4 °C with appropriate primary antibodies, subsequently with specific secondary antibodies and visualized using the enhanced chemiluminescence reagent (Thermo Scientific). The following antibodies were used: Rabbit anti-alpha-TUBULIN, mouse anti-E-cadherin, and rabbit anti-NF-κB p65 were from Abcam; rabbit antibodies against p38, phospho-p38, JNK, phospho-JNK, ERK, phospho-ERK, MLC, phosphor-MLC, phospho-NF-κB p65, STAT1, and phospho-STAT1 (Tyr701) were from Cell Signalling Technology; mouse anti-CLDN-2 and rabbit anti-ILDR-1 were from Invitrogen; anti-mouse/rabbit IgG horseradish peroxidase-linked secondary antibodies were from Cell Signalling Technology. Original western blot images were included in Supplementary Figures 7, 8, and 9. Densitometric quantification analyses of the western blots were performed using the Image J software.