Ab3471

Lysates of cultured cardiac fibroblasts and tissues lysates of infarcted hearts were extracted using a lysis buffer (Beyotime, Shanghai, China) with a protease inhibitor cocktail (Roche, Germany). Protein blotting was performed with a modified standard protocol using primary antibodies to VPO1 (1:1000, ABS1675, Millipore, USA), α-SMA (1:1000; ab7817, Abcam, UK), collagen I (1:1000; ab3471, Abcam, UK), smad2/3 (#8685), P-smad2/3 (#9510), ERK1/2 (#4695), P-ERK1/2 (#4370) (1:1000; Cell Signaling Technology, USA), 3-chlorotyrosine (1:500; HP5002, Hycult Biotech, Netherlands), GAPDH (1:5000, G8795, Sigma-Aldrich, USA) overnight at 4 °C. PVDF membranes were incubated with HRP--conjugated secondary antibodies and bands were visualized by a gel documentation system (Bio-Rad, USA).

Human and mouse heart tissues were fixed overnight in 4% paraformaldehyde. Samples were embedded in paraffin wax and sliced into 4 μm sections. Masson trichrome stain (Sigma-Aldrich, USA) was used according to the manufacture's protocol. Infarct size, expressed as a percentage, was calculated by dividing the sum of infarct areas from all sections by the sum of LV areas from all sections (including those without infarct scar) and multiplying by 100 [14 (link)]. Infarct expansion index was calculated using the following formula: expansion index = (LV cavity area/total heart area) × (uninfarcted septal thickness/infarcted LV free wall thickness) [15 ]. Rabbit anti-VPO1 (1:200; ABS1675, Millipore, USA), anti-3-chlorotyrosine (1:100; HP5002, Hycult Biotech, Netherlands), anti-collagen I (1:200; ab3471, Abcam, UK), anti-ki67 (1:200, ab16667, Abcam, UK), and mouse anti-α-SMA antibody (1:200; ab7817, Abcam, UK) were used as primary antibodies for immunochemical staining, followed by secondary antibodies (1:100; ZSGB-BIO, China) and DAB reagents. Quantification of fibrosis, VPO1, α-SMA and collagen I in heart tissues was determined by Imag-Pro-Plus 6.0 software.

For immunofluorescence assays, cells cultured into alginate scaffolds were fixed with 4% paraformaldehyde for 1 hour at RT followed by two washes with PBS1X for 5 minutes and permeabilization with 0.5% Triton X‐100 in PBS for 20 minutes. For blocking unspecific binding, alginate scaffolds were incubated in 3% BSA in PBS for 20 minutes and washed twice with PBS 1X for 5 minutes. Scaffolds were incubated with the primary antibodies for 48 hours at 4°C. Primary antibodies were a rabbit monoclonal antibody against RUNX2 (DIL7F, Cell Signaling Technology, Beverly, MA, http://www.cellsignal.com) and a rabbit polyclonal against Collagen I (ab3471, Abcam, Cambridge, U.K., http://www.abcam.com). After incubation with the primary antibodies, samples were washed and incubated with an anti‐rabbit IgG conjugated with FITC (Jackson InmunoResearch Laboratories, 11‐095‐045) for 1 hour in the dark. Nuclei were detected with DAPI‐like Hoechst. Scaffolds were mounted in Prolong Gold (Invitrogen) and samples analyzed 24 hours later.