The following primary monoclonal antibodies were used for an immunocytochemical investigation: type I collagen (1:150, Abcam), elastin (1:50, Abcam), AP-1 (1:200, Sigma), Klotho (1:250, Abcam), MTH-1 (1:1000, Abcam), melatonin receptor 1A (1:200, Abcam), melatonin receptor 1B (1:100, Abcam), clock (1:100, Abcam), cytokeratin 20 (1:150, Dako). The secondary antibodies conjugated with Alexa Fluor 488 and 647 (1:1000, Abcam) were used for immunofluorescent microscopy. Confocal microscopy was carried out using an Olympus FV 1000 microscope (Japan).
Since the investigation was concerned with unique dermal Merkel cells, to confirm the existence of a subpopulation of these cells in the culture, we used a special marker cytokeratin-20. We applied the method of double labeling using antibodies to cytokeratin-20 (green fluorescence) and antibodies to Klotho (red fluorescence). The area of expression was calculated in relation to Merkel cells that were immunopositive to CK-20 cells.
Since the investigation was concerned with unique dermal Merkel cells, to confirm the existence of a subpopulation of these cells in the culture, we used a special marker cytokeratin-20. We applied the method of double labeling using antibodies to cytokeratin-20 (green fluorescence) and antibodies to Klotho (red fluorescence). The area of expression was calculated in relation to Merkel cells that were immunopositive to CK-20 cells.