Ab2057181

Formalin-fixed, paraffin-embedded intestinal (cecal, colon and ileum) tissue section (4 μm) were incubated overnight at 4 °C with a primary antibody (Table 3) after antigen retrieval according to the manufacturer’s instructions.Table 3

Information of antibodies

Name of antibody	Company name and Catalog no.	Dilution factor
Anti-Mast Cell Tryptase antibody [AA1]	abcam, ab2378	1:1000
Anti-IL-6 antibody [MP5-20F3]	abcam, ab191194	1:100
MUC2 Antibody	Novus, 31231	1:100
Goat Anti-Rabbit IgG H&L (HRP)	abcam, ab205718	1:1000
Goat Anti-Mouse IgG H&L (HRP)	abcam ab97023	1:2500
Recombinant Human GDF11 protein (ab50159)	abcam ab50159	1:1000

Samples were washed and subsequently incubated with secondary antibody for 45 min at RT (Table 3) Sections were counterstained either with the diaminobenzidine substrate kit (Nacalai, USA) followed by hematoxylin, or sections were stained with 6-diamidino-2-phenylindole (DAPI, Thermo Fisher, USA) for visualization of cell nuclei as previously described.

Cells were lysed with radio-immunoprecipitation assay lysis buffer (APExBIO, K1020), supplemented with protease inhibitor cocktail (APExBIO, K1007) and phosphatase inhibitor cocktail (APExBIO, K1015). Insoluble cell debris was removed by 15-min centrifugation at 13,500×g at 4 °C. Protein concentration was determined using a bicinchoninic acid protein assay kit (Beyotime, P0010S). Western blot was performed on total protein extracts. Proteins were then transferred to 0.45-μm polyvinylidene difluoride membranes (Sigma, IPVH00010) after electrophoresis. Membranes were blocked for 1 h at room temperature in blocking buffer (Beyotime, P0023B) and incubated overnight at 4 °C with antibodies in primary antibody dilution buffer (Beyotime, P0023A). Goat-anti-Rabbit horseradish peroxidase-conjugated (Abcam, ab2057181:10,000) antibody was used as secondary antibody. Protein bands were visualized with Clarity Western ECL Substrate (Bio-Rad, USA) and the pictures were obtained by ChemiScope Western Blot Imaging System (Clinx Science Instruments, China).
Primary antibodies and corresponding concentrations were used as follows: rabbit monoclonal anti-SUN1 (Abcam, ab124770, 1:1000) and rabbit monoclonal anti-SUN2 (Abcam, ab124916, 1:1000). Glyceraldehyde 3-phosphate dehydrogenase (Abcam, ab8245, 1:1000) was used as a loading control.