Imagej

Cryosections of the heart auricle tissue or cultivated cells were fixed for 20 min using 4% paraformaldehyde (PFA), washed and permeabilized in PBS with TritonX-100 (tissue: 0.2%, cells: 0.02%, Applichem, Darmstadt, Germany) and supplemented with 5% goat serum for 30 min. The applied primary antibodies were diluted in PBS as followed: mouse anti-Nestin 1:200 (Millipore, Burlington, MA, USA), rabbit anti-S100B 1:500 (Dako, Glostrup, Denmark), rabbit anti-α-actinin (Cell-Signaling, Danvers, MA, USA), mouse anti Connexin 43 (Millipore). They were applied for 1 h (cells) or for 2 h (sections), both at room temperature (RT). After three washing steps, secondary fluorochrome-conjugated antibodies (Alexa 555 anti-mouse or Alexa 488 anti-rabbit, Invitrogen, Life Technologies GmbH, Carlsbad, CA, USA) were applied for 1 h at RT with a dilution ratio of 1:300. Nuclear staining was realized by incubation with 4,6-diamidin-2-phenylindol (DAPI) (1 μg/mL, Applichem) in PBS for 15 min at RT. Finally, the samples were mounted with Mowiol (self-made). Imaging was performed using a confocal laser scanning microscope (CLSM 780, Carl Zeiss, Oberkochen, Germany) and image processing was executed with ImageJ and CorelDRAW [25 (link)] (open source and Corel Corporation).