Ab108508

The ileal tissue sections were processed (Tissue-Tek^® VIP^®; Sakura Finetek) and fixed in paraffin wax. The wax blocks were cut into 5-µm-thick slices and deparaffinized. Antigen retrieval was performed in sodium citrate buffer. The sections were blocked in 5% bovine serum albumin (BSA) in PBS for 1 h, and then incubated with the primary antibody overnight at 4 °C. Subsequently, the sections were processed using the DAB Detection Kit (SP-9000-D; ZSGB-Bio, Beijing, China) according to the manufacturer’s protocol. Hematoxylin staining was used to counterstain nuclei. The IHC features shown in the figures are representative of all tissue samples studied. The primary antibody are as following: lysozyme (ab-108508; Abcam; 1:1000 dilution); IAP (ab-108337; Abcam; 1:250 dilution).

At least four consecutive sections of the same jejunum tissue were selected; after deparaffinization of jejunum sections, antigen retrieval was performed with 10 mM pH 6.0 sodium citrate solution in a 95 °C water bath for 10 min, followed by a 20 min incubation at room temperature. Slides were washed, blocked in 5% normal goat serum at 37 °C, and stained using the primary antibodies rabbit anti-lysozyme antibody (1:200, ab108508, Abcam, Cambridge, CA, USA) and rabbit anti-NLRP6 antibody (1:1000, 144-61128-50, Raybiotech, Shanghai, China) overnight at 4 °C, respectively. Then, rabbit anti–lysozyme antibody-stained sections were incubated with goat anti-rabbit Alexa Fluor 594 (1:300, ab150080, Abcam, Cambridge, CA, USA), and rabbit anti-NLRP6 antibody-stained sections were incubated with goat anti-rabbit Alexa Fluor 488 (1:400, ab150077, Abcam, Cambridge, CA, USA). The sections were photographed with a Nikon Eclipse TE 2000S inverted microscope (Nikon Instruments Co., Inc., New York, NY, USA). The same position of the serial sections stained with the two antibodies were photographed and counted, respectively. The numbers of positively stained puncta were counted using Image-Pro Plus software (Media Cybernetics, Rockville, MD, USA).

For all histological endpoints in small intestinal tissues, the samples were fixed in 10% neutral buffered formalin, embedded in paraffin, and sectioned at 5 μm. Tissue sections were dewaxed, rehydrated, and then stained. Lysozyme analysis of small intestinal sections was done by immunohistochemistry (HRP/DAB (ABC) IHC Detection Kit, Abcam, UK), following manufacturer recommendations. Anti-lysozyme antibody (ab108508) was purchased from Abcam, UK. Images were captured by microscopy (Olympus, Tokyo, Japan). The DAB staining intensity was analyzed using ImageJ (1.8.0_172) and represented by the average optical density (AOD) value (AOD = IOD/area, IOD: integrated option density). We examined five successive fields as a section, and three sections per mouse were used for analysis. The evaluation of staining intensity was performed by two independent assessors.

The frozen sections were warmed at room temperature after being taken from the − 20℃ fridge. Samples were washed and permeabilized with Triton X100 (0.1%) in PBS for 30 min, blocked with goat serum (10%) at RT for 1 h, and incubated with primary antibody at 4℃ overnight. The next day, after washing, the samples were incubated with a secondary antibody at 37℃ for 1 h in darkness, counterstained with mixed DAPI & Mounting solution (1:1), and mounted for observation (Nikon ECLIPSE 80i). The following antibodies were used: αSMA (1:100; Sigma; A5228) and lysozyme (1:200; Abcam; ab108508). CRS4C (1:100) was generated in Dr. Junping Wang’s lab.

Animals were perfused with phosphate-buffered saline (PBS) and 4% paraformaldehyde (PFA) in PBS. Small intestine was fixed in 4% PFA overnight and stored in 30% sucrose. A small piece of small intestine embedded in optimal cutting temperature (OCT) compound and sectioned with 7 -µm thickness for immunofluorescence. Tissue sections were placed on glass slides and washed with PBS for 5 min. 1% sodium dodecyl sulfate in PBS was used for antigen retrieval for 5 min. and then tissues were placed in blocking solution (mixture of 3% bovine serum albumin, 10% goat serum, and 0.1% triton-X 100 dissolved in PBS) for 1 hour. Tissue was treated with for anti-lysozyme antibody (Abcam (ab108508), 1:50 in blocking solution) overnight and 1 hour with the secondary antibody (Alexa594, Life Science, 1:100 in blocking solution).

Rabbit anti-Muc2 (1:200, sc-15,334; Santa Cruz), rabbit anti-ChgA (1:200, ab15160; Abcam), rabbit anti-Ki67 (1:200, ab15580; Abcam), rabbit anti-Lyz (1:200, ab108508; Abcam), mouse anti-E cadherin (1:200, 610,182; BD Biosciences), rabbit anti-Ca7 (1:200, 13,670–1-AP; Proteintech), rabbit anti-Ces1(1:200, 16,912–1-AP, Proteintech), rabbit anti-Ces2 (1:200, ab184957, Abcam).