For immunofluorescence, heart sections (8 μm) were incubated with primary antibodies against CD68 (Serotec, 1:200), CD301 (Biolegend, 1:200), PCNA (CST, 1:800), CD31 (RD, 1:200), VEGF (Proteintech, 1:200), SMA (Sigma, 1:500), CollagenΙ(Proteintech,1:200), THBS1(Proteintech, 1:200) and Alexa Fluor 488/594/633-conjugated secondary antibodies (Invitrogen, Carlsbad, CA, 1:1,000) for 1 h at room temperature, respectively. The embedded hearts were washed and stained with DAPI (Millipore, MA). Images were captured under a Olympus (FV1000) laser-scanning confocal microscope from each heart section for further analysis. Positive staining area and relative mean fluorescent intensity were measured using Image-Pro Plus software 6.0 (Media Cybernetics, Rockville, MD, USA)50 (link).
For immunocytochemistry, primary Mos/Mps grown on slides were stimulated with or without PGE2 and then fixed with 4% paraformaldehyde. The slides were blocked in TBST containing 1% bovine serum albumin after washing, incubated with primary antibodies against CD68 (Serotec, 1:200) or VEGF (Proteintech, 1:200) overnight at 4 °C, stained with a secondary antibody for 2 h at room temperature, and photographed using fluorescent microscopy as described above. At least five random images were taken in region of each slide and positive signalling was quantified as previously described51 (link).
For immunocytochemistry, primary Mos/Mps grown on slides were stimulated with or without PGE2 and then fixed with 4% paraformaldehyde. The slides were blocked in TBST containing 1% bovine serum albumin after washing, incubated with primary antibodies against CD68 (Serotec, 1:200) or VEGF (Proteintech, 1:200) overnight at 4 °C, stained with a secondary antibody for 2 h at room temperature, and photographed using fluorescent microscopy as described above. At least five random images were taken in region of each slide and positive signalling was quantified as previously described51 (link).