Picro sirius red staining

Atria were fixed and sectioned for histological analysis of inflammatory infiltrates by staining for hematoxylin and eosin (MilliporeSigma), activated T lymphocytes (CD3, ab16669, Abcam; CD4, ab237722, Abcam), macrophage infiltration/polarization (CD68, ab125212, Abcam; CD163, ab182422, Abcam), and neutrophil infiltration (CD11b, ab133357, Abcam). hematoxylin and eosin staining was quantified using the ImageJ color deconvolution plugin (48 (link), 71 (link)). Wheat germ agglutinin (Thermo Fisher Scientific), which selectively binds the cell membrane of mammalian cells, was used to identify cell borders. Left atrial collagen was quantified based on hydroxyproline content measurement (K555-100, BioVision) (72 (link), 73 (link)). Atrial fibrosis was verified using Masson’s trichrome (Thermo Fisher Scientific) and picrosirius red staining (Thermo Fisher Scientific) (21 (link), 49 (link), 50 (link)).

The harvested constructs were cut into two parts; one half was embedded in polymethylmethacrylate (MMA, Sigma-Aldrich, Germany) and the other half was slowly decalcified in formalin and ethylenedi-amine tetra-acetic acid (EDTA, Sigma-Aldrich, Germany) for 3 months and subsequently embedded in paraffin. Samples embedded in MMA were sectioned in 300–400 μm slices using a Leica 4 SP1600 Saw Microtome system (Leica, Germany). After sectioning, the samples were stained with methylene blue/basic fuchsine and evaluated with light microscopy (Olympus BX51, Japan) [34 (link)]. Paraffin-embedded samples were sectioned using a microtome (n = 6) (Leica sawing microtome, Nusslochh, Germany) in 5 μm slices and stained with hematoxylin and eosin (H&E staining, thermo Fisher scientific, USA) for tissue overview analysis, and picro-sirius red staining (Thermo Fisher Scientific, USA) for collagen analysis. Collagen orientation was visualized with polarized light (Olympus BX51, Japan). Backscatter images using a Secondary Electron Detector were analyzed by EDX with a scanning electron microscope (FEI XL30SFEG, USA). Before EDX analysis for newly formed bone and native bone, MMA sections were polished and sputtered with gold.