Immunohistochemical Analysis of Paraffin-Embedded Cardiac Tissue

Paraffin sections (2 µm) were processed for immunohistochemistry [28 (link)]. Sections were deparaffinized with Roti^®-Histol (Carl Roth, Karlsruhe, Germany 6640) and hydrated by ethanol gradient (100–40%). H&E staining was performed by firstly adding hematoxylin solution (Sigma-Aldrich, GHS316) for 45 s followed by 10 s tap water and incubation of Eosin (Sigma-Aldrich, HT110232) for 1 min. After staining, samples were mounted with Entellan^® (VWR, Radnor, PA, USA 1079610500). Whole heart sections were scanned using MIRAX Scanner from Zeiss (Oberkochen, Germany). Quantification of stained area (cell size) was performed using software MIRAX Viewer and ZEN 2 (blue edition) both from Zeiss.