Alexa fluor 488 or 633

Deparaffinization and epitope retrieval procedures of the paraffin-embedded liver and small intestine sections were carried out in an exactly similar manner as mentioned for the immunohistochemistry method. After the epitope retrieval process was completed, all tissue sections were permeabilized using PBS-Tx (PBS+ 0.1% Triton X-100) solution for 1 h, followed by blocking with 5% goat serum for 1 h. Following the blocking step, the tissue sections were probed with anti-Claudin2, anti-Occludin, anti-NLRP3, anti-ASC2, anti-Smad2/3, and anti-Smad4 primary antibodies (1:300 dilution) and kept overnight at 4 °C in a humidified chamber. After that, species-specific anti-IgG secondary antibodies conjugated with Alexa Fluor 488 or 633 from Invitrogen (Rockford, IL, USA) were applied onto the sections (1:250 dilution). Lastly, ProLong Gold antifade reagent with DAPI (Life Technologies, Carlsbad, CA, USA) was used to mount the tissue sections. All immunofluorescence-stained images for this study were captured by an Olympus BX63 microscope (Olympus America, Center Valley, PA, USA) using the 40× objective. Analyses of all morphometric data were performed using CellSens Software from Olympus America (Center Valley, PA, USA).

Deparaffinization and epitope retrieval procedures of the paraffin-embedded small intestine and brain tissue sections were performed similarly as mentioned above. After completion of the epitope retrieval process, the tissue sections were permeabilized by using PBS-T (PBS+ 0.1% Triton X-100) solution for 1 h, followed by blocking with 5% goat serum for 1 h. After that, tissue sections were incubated with primary antibodies of anti-Occludin, anti-Claudin-2, anti-TLR4, anti-Flotillin1, anti-Claudin-5, anti-CD31, anti-CD40, and anti-TMEM119 (at 1:300 dilution) and kept overnight at 4 °C in a humidified chamber. Then, species-specific anti-IgG secondary antibodies conjugated with Alexa Fluor 488 or 633 from Invitrogen (Rockford, IL, USA) were used at 1:250 dilutions. Finally, mounting was performed by using ProLong Gold antifade reagent with DAPI (Life Technologies, Carlsbad, CA, USA). Images were taken under 10×, 40×, and 60× magnification with an Olympus BX63 microscope. Morphometric data analyses were performed by CellSens Software from Olympus America (Center Valley, PA, USA).

Cells plated onto Labtek™ slides were fixed in 4% paraformaldehyde and permeabilized in 0.2% Triton X-100 in PBS for 10 min each. After washing in PBS, cells were incubated overnight at 4 °C with rabbit polyclonal anti-p52 or anti-RelB antibody (1:100), followed by incubation with anti-rabbit Alexa Fluor 488 or 633 (1:300, Invitrogen). After washing, cells were mounted in 70% glycerol in PBS, and analyzed with a DM-IRB confocal microscope (Leica DM, Bannockburn, IL)31 (link). Nuclei cells were stained with DAPI (Vector Laboratories, Inc., Burlingame, CA) to observe the typical morphological changes.