Ab109226

The intestinal tissues were homogenized in a RIPA extraction buffer with a protease inhibitor cocktail (Roche) for 30 min. Proteins were quantified via a BCA protein assay kit (Beyotime Biotechnology, China), and then prepared in Laemmli buffer and boiled in water bath. Equal amounts of tissue lysates (50 mg) were loaded and separated on10% SDS-PAGE gels with constant voltage of 100 V for 100 min, and transferred to PVDF membranes with constant current of 300 mA for 80 min in ice bath. Membranes were subsequently blocked in 8% (w/v) non-fat dried milk for 1 h at room temperature, then incubated overnight at 4 °C with specific antibodies against tryptase (Abcam, ab109226, 1:2000), PAR2 (Abcam, ab154835, 1:1000), β-actin(Abbkine, A01010, 1:2000) and GAPDH (Abbkine, A01020, 1:2000). After fully washing, the membranes were probed with HRP-conjugated goat anti-rabbit or mouse antibody (1:5000, Pierce, USA) for 1 h at room temperature. Then, the protein bands were developed in the SuperSignal West Pico Substrate (Pierce, USA) and quantified by the FluorChem FC3 software (ProteinSimple, USA).

Immunofluorescence was performed as protocol (Im et al., 2019 (link)). Coronal paraffin-embedded slices were blocked and incubated with primary antibodies as follows: anti-6E10 (1:1,000, 803001, Biolegend), anti-CHRM1 (1:100, BA 1543, Boster), anti-CHRM2 (1:100, ab 109226, Abcam), anti-Doublecortin (DCX, 1:500, ab135349, Abcam), and anti-Neuro-D1 (1:200, ab60704, Abcam) overnight in 4°C. Anti-mouse 488, anti-mouse 555, and anti-rabbit 555 (1:1,000, Thermo Fisher Scientific) were used and nuclei were visualized with DAPI. Sections of each mouse were taken to acquire images in MS/VDB and DG at high power magnification (200×) by confocal microscope. Images were analyzed by Image J and results are expressed as positive areas or cells. Negative controls were processed in every immunofluorescence run.