Dab color developing solution

Jejunum and ileum sections were fixed in 4% paraformaldehyde and embedded in paraffin as described [22 (link)]. Mast cells in the jejunum were visualized using the Naphthol AS-D chloroacetate esterase staining kit (Sigma-Aldrich). Eosinophils in the jejunum and Foxp3⁺T cells in the ileum were identified by immunohistochemistry (IHC) by incubating tissue sections with anti-mouse major basic protein monoclonal antibody (1:1500; provided by Jamie J. Lee, Mayo Clinic, Scottsdale, AZ, USA) and anti-mouse/rat Foxp3 monoclonal antibody (clone FJK-16s; 1:100; eBioscience), respectively. The sections were then developed with HRP conjugated goat anti-rat IgG antibody (Millipore, Billerica, MA, USA), DAB color-developing solution (Dako, Glostrup Municipality, Denmark), and counter-stained with hematoxylin gill number 3 (Sigma-Aldrich). For all analyses, two tissue sections were stained. Quantification of mast cells and eosinophils was performed by analyzing three random sections per stained sample on the ImageJ software and expressed as number of cells per mm² of mucosa.

To detect NLRP3 expression in tissues, the paraffin-embedded liver tissue block was sliced into 4 μm serial sections, deparaffinized in xylene, and treated with gradient ethanol, followed by antigen retrieval. The endogenous peroxidase was eliminated by 3% hydrogen peroxide. Later, sections were blocked with 2% bovine serum albumin (BSA) at 37° C. After non-specific antigen-antibody binding, sections were incubated with anti-NLRP3 monoclonal antibody (dilution, 1:250; Abcam, MA, USA) and subsequently with peroxidase-labeled streptomycin (Abcam, MA, USA) for 15 min. Afterwards, each section was added with drops of freshly prepared DAB color developing solution (DAKO, Denmark), sufficiently washed with tap water, countered-stained with hematoxylin, and mounted.