Cardiac Tissue Preparation and Fixation

Animals were killed by cervical dislocation and hearts were arrested in diastole using a 0.1 mol L⁻¹ cadmium chloride solution (Sigma, Gillingham, UK) before removal from the chest cavity. Whole hearts were weighed, washed briefly in PBS before 24 h fixation in formalin (Sigma) then stored for 72 h in 70% ethanol. The atria were removed and the ventricles dissected into the apex, mid, and base regions and all tissue was processed for paraffin embedding using a Leica TP1020 tissue processor as previously described (Waring et al. 2014 (link)). Five micrometer tissue sections were cut using a Leica RM2235 microtome, mounted onto polysine microscope slides (ThermoFisher, Loughborough, UK), and stored at room temperature until processing for immunohistochemistry.