Histologic evaluation was performed on paraffin sections of two to three EMBs using standard procedures. In brief, formaldehyde-fixed biopsies were embedded in paraffin, cut into 4 µm thick slices and stained with Trichrome stain AB solution (Sigma, Taufkirchen, Germany), Bouin’s solution (Sigma, Taufkirchen, Germany) and Weigert A&B (Morphisto GmbH, Frankfurt, Germany). Quantification of fibrous connective tissue was performed using ImageJ 1.53A software. Active myocarditis was excluded according the histomorphologic Dallas criteria [
1]. Immunohistochemistry was used to quantify inflammatory infiltrates and was carried out on RNAlater-fixed EMBs. Specimens were embedded in Shandon™ Cryomatrix (Thermo Fisher Scientific, Waltham, MA, USA), cut serially into cryosections of 5 µm thickness and placed on 10% poly‐L‐lysine‐precoated slides. Myocardial inflammation was assessed referring to the 2013 ESC statement [
6]. In detail, inflammatory cardiomyopathy was diagnosed as CD3
+ T-lymphocytes ≥ 14 cells/mm
2 (1:700, Dako, Glostrup, Denmark), CD45R0
+ T-memory cells ≥ 50 cells/mm
2 (1:300, Dako, Glostrup, Denmark), CD11a
+/LFA-1
+ lymphocytes ≥ 14 cells/mm
2 (1:250, Immuno Tools, Friesoythe, Germany), CD11b
+/Mac-1
+ macrophages ≥ 40 cells/mm
2 (1:500, ImmunoTools, Friesoythe, Germany) and/or perforin
+ cytotoxic cells ≥ 2.9 cells/mm
2 (1:150, BD Bioscience, San Jose, CA, USA). Tissue and endothelial activation were measured by the expression of the intracellular adhesion molecule ICAM-1 (1:800, Immuno Tools, Friesoythe, Germany). As a secondary antibody, EnVision peroxidase‐conjugated anti‐mouse/anti-rabbit antibody (Dako, Glostrup, Denmark) was applied. Immunohistochemical staining was visualized using 3‐amino‐9‐ethylcarbazole as chromogenic substrate (Merck, Darmstadt, Germany). Finally, slides were counterstained in hematoxylin and mounted with Faramount aqueous mounting medium (Dako, Glostrup, Denmark). All markers were quantified using quantitative digital-imaging analysis as described before [
11]. PAI-1 expression was analyzed on EMB cryosections using an appropriate antibody (1:1.000, Gentaur, Kampenhout, Belgium). PAI-1 expression area was quantified and depicted as percent of analyzed area (area fraction %). Quantification of myofibroblasts was performed on paraffin-embedded sections using an anti-alpha smooth muscle actin (α-SMA) antibody (1:200, Cell Signaling, Danvers, MA, USA). α-SMA
+ cells were counted using ImageJ 1.53A software and are depicted as cells/mm
2. TGF-β levels were quantified on paraffin-embedded sections using an anti-TGF-β antibody (1:1.000, antibodies-online GmbH, Aachen, Germany) and are depicted as percent of analyzed area (area fraction %).