Ab86482

Total protein of colonic tissues was extracted according to the manufacturer’s protocol (Vazyme, USA). Briefly, protein concentrations were determined through BCA Protein Assay Kit (Vazyme, USA). Samples with equal amounts of protein (25 µg) were fractionated on 10% SDS polyacrylamide gels, transferred to polyvinylidene difluoride (PVDF) membranes, and blocked in 5% skim milk in TBST for 1.5 h at 25°C. The membranes were then incubated at 4°C overnight with 1:1,000 dilutions (v/v) of the primary antibodies. After washing the membranes with TBST, the secondary antibodies with 1:1,000 dilutions were added and incubated for another 2 h at 25°C. Protein expressions were examined using an Enhanced Chemiluminescence Detection System. GAPDH was used as a loading control. Antibodies in western blotting were purchased from Abcam (Cambridge, MA, USA), including Col1a2 (ab96723), Col3a2 (ab196613), MMP-1 (ab52631), MMP-3 (ab52915), TIMP-1 (ab86482), E-cadherin (ab76055), N-cadherin (ab202030), Vimentin (ab193555), a-SMA (ab32575), MMP-9 (ab38898) and GAPDH (ab181602).

Liver samples were fixed with PBS 4% PFA, paraffin embedded and sectioned (4 μM). Slides were de-paraffinized using Wcap solution (Bio optica, Milano, Italy) (75°C, 20 min). Antigen retrieval was performed in 0.1 M EDTA, pH 8.0 (Diagnostic BioSystems, CA, USA) using a pressure cooker (125°C, 3 min), followed by washes with worm ddH₂O. Samples were blocked in PBS, 20% normal horse serum, and 0.2% Triton X-100 (20 min, 25°C) and then incubated with primary Ab in PBS, containing 2% normal horse serum and 0.2% Triton X-100 (60 min, 25°C). Next, samples were washed three times in PBS and incubated with a secondary antibody (60 min, 25°C) and mounted in a mounting medium. Primary Abs: anti-GFP antibody (ab6673, Abcam), anti MMP-14 (ab51074, Abcam), TIMP1 (ab86482, Abcam). For MMP-14 staining, the samples were incubated with a biotin antibody (711-065-152, Jackson ImmunoResearch) following the incubation of the first Ab. Then, a cy3-streptavidin (016-160-084, Jackson ImmunoResearch) was used as a secondary Ab. TIMP1 staining was done following in situ zymography after the sections were fixed with 4% PFA.