Fig. 4
Immunocytochemical analysis of cells dispersed from pacemaker cell clusters (PCC). Signals were visualized by confocal (a) and fluorescence microscopy (b). Cells were immunolabeled and imaged by using identical protocols. a Left column (from top to bottom): anti-HCN1, anti-HCN4, anti-NCX, and anti-Cav1.2 staining indicates distinct membrane expression of the respective proteins. Note that anti-SHOX2 immunolabeling is concentrated to the nucleus. Middle column: nuclei of respective samples are counterstained with propidium iodide (PI). Right column: overlay of immunostains and PI counterstain. b Fluorescence microscopic visualization of anti-Cx45, anti-NCX, and anti-Cav1.2 in less dispersed areas of PCC, showing abundant membrane expression. In contrast, anti-cTnI immunolabeling reveals only little signal intensity, while images of other working-type cardiomyocyte marker proteins display no specific membrane signal (Cx43, Nav1.5). Nuclei are counterstained with PI (red). Scale bars = 20 μm. cTnI cardiac troponin I, Cx connexin, HCN hyperpolarization-activated cyclic nucleotide channels, NCX sodium calcium exchanger