Tryptase

Paraffin-embedded intestine blocks were sliced into 4 μm sections. Tryptase (1 : 500, Santa Cruz Biotechnology, Dallas, TX, USA) staining was carried out to evaluate the expression of Tryptase in MCs by using immunofluorescence methods. Sections were incubated with the Tryptase primary antibody overnight at 4°C. A secondary antibody (1 : 200, Life Technologies, Carlsbad, CA, USA) was added for 1 hour at 37°C, and then the sections were rinsed with PBS three times. A microscope (DMLB2, Leica, Wetzlar, Germany) was used to observe the stained sections.

Immunoblot and immunoprecipitation were performed according to the standard procedures (Kim et al., 2018 (link)). The following primary antibodies were used: anti-HDAC6, TLR2, TLR4, and SIRT1 (ABclonal); anti-FcεRIβ, Lyn, GATA3, T-bet, JNK1, pJNK1^T183/Y185, Tryptase, Chymase, BECN1, MyD88, TSG101, and Calnexin (Santa Cruz Biotechnology); anti-CXCL13 (R&D Systems); anti-CD163, FoxP3, TSLP, and MIP-2 (Abcam); anti-iNOS, pBECN1^S14, COX2, ERK1/2, pERK^T204, HDAC3, NFκB, AMPKα, pAMPKα^T172, IKBα, pIKBα^S32, p38MAPK, p-p38MAPK^T180/Y182, and LC3(Cell Signaling Technology). The detailed information of primary antibodies is described in Supplemental Table S2.To isolate tissue lysates, tissue was frozen in liquid nitrogen to preserve protein structure and homogenized with RIPA buffer. After lysis, vortexing and centrifugation at 10,000 X g for 15 min at 4°C were followed. Supernatant was then obtained and used as tissue lysates for immunoblot and immunoprecipitation.

There are two major types of immunofluorescent staining method: (1) Cells that attached to coverslips after experimental treatment were fixed with 4% paraformaldehyde for 20 min, and permeability with 0.2% Triton X-100 and blocked with PBS containing 5% BSA for 60 min in the next. The coverslips were followed by incubating with primary ZO-1/CRHR1/CRHR2, and labeled with specific secondary antibody for 1 h, and stained with DAPI (Biosharp, Hefei, China) for 5 min. The slides were washed with PBS and mounted by FluorSave^TM mounting media (Merck, Darmstadt, Germany). (2) The intestinal tissue was fixed in 10% phosphate-buffered formalin for immunofluorescent studies before paraffin embedding. For immunofluorescent staining of ZO-1 (1:500, Proteintech, Wuhan, Chian), CRHR1 (1:50, Proteintech, Wuhan, Chian), CRHR2 (1:200, Proteintech, Wuhan, Chian) and Tryptase (1:100, Santa Cruz, CA, USA), paraffin sections (5 μm) were dewaxed to rehydrate. After blocking with 10% goat serum, sections were incubated with primary antibody at 4 °C overnight, and detected with IgG (H + L) Highly Cross-Adsorbed Secondary Antibody (Thermofisher, Shanghai, China) for 1 h at room temperature, respectively. Representative images were captured under a fluorescence microscope and analyzed with imageJ Software (ImageJ 1.46r, National Institutes of Health, Bethesda, MD, USA).