Ab166898

Tissues or cells were fixed with 4% paraformaldehyde, stabilized in 0.2% Triton X-100 for 10 min until cell membrane rupture, washed with PBS 3 times, and immersed in 2% BSA for 30 min to inhibit nonspecific antigen binding sites. The tissues were then incubated with anti-trypsinogen (1:500, Abcam, Cambridge, UK, ab166898) antibodies and anti-alpha smooth muscle actin (1:500, Abcam, Cambridge, UK, ab124964) overnight at 4 °C. The cells were then incubated with anti-trypsinogen (1:500, Abcam, Cambridge, UK, ab166898) antibodies or anti-alpha smooth muscle actin (1:500, Abcam, Cambridge, UK, ab124964) overnight at 4 °C. After washing, the tissues or cells were incubated with secondary antibody (Invitrogen) for 60 min, and the nuclei were stained with DAPI (Invitrogen) for 2 min. Then, the cells were washed with PBS and shielded from light before observation with a fluorescence microscope.

These experiments were performed as we previously described^{21 (link)–23 (link)}. For Western blot, antibodies for GAPDH (sc-32233; Santa Cruz Biotechnology, Santa Cruz, CA, USA), Cosmc (sc-67480; Santa Cruz Biotechnology), CEL (ab79131; Abcam, Cambridge, UK; sc-34883; Santa Cruz Biotechnology), DMBT1 (MAB59151; R&D Systems, Minneapolis, MN, USA), elastase (ab21593; Abcam) and trypsin (ab166898; Abcam) were used. Ten micrograms per milliliter of Vicia villosa lectin (VVA) reactive against O-GalNAc (Tn antigen) (B-1235; Vector Laboratories, Burlingame, CA, USA) complexed with 1 µg streptavidin-HRP (21126; Pierce, Thermo Fisher Scientific, Grand Island, NY, USA) was used. For immunohistochemistry, antibodies for Tn Antigen (MA180055; Thermo Fisher Scientific, Grand Island, NY, USA), STn Antigen (ab115957; Abcam), insulin (8138; CST, Beverly, MA, USA) and VVA-fluorescein (FL-1231; Vector Laboratories) were used at a dilution of 1:100^{23 (link)}. Ten micrograms per milliliter of Sambucus nigra lectin (SNA) (B- 1305–2; Vector Laboratories, Burlingame, CA, USA) was complexed with 1 µg streptavidin-HRP. For real-time PCR, RNA was extracted with the RNeasy Plus Tissue Mini Kit (Qiagen).