Indirect Immunostaining on Mouse Embryonic Heart for the Detection of Proliferated Cardiomyocyte

The heart is the first organ to form and become functional in a developing embryo and its proper function is critical for most, if not all, subsequent stages of an animal's development. The formation of the heart relies heavily upon the rapid proliferation of cardiomyocytes at a specific stage during early development, and interfering with the ability of these cells to grow during this time frame results in heart defects that include thin ventricle walls, as well as malformed ventricular septums. Thus, since cardiomyocyte proliferation represents a key step in early cardiac development and identifying the mechanisms that underlie cardiomyocyte proliferation has become an important area of study, techniques to identify and study proliferating cardiomyocytes in vivo are needed. Immunofluorescence and immunohistochemistry staining are powerful tools for studying cardiomyocyte proliferation in the developing animal. The phosphorylation of histone H3 at Ser10 (pH3) only occurs in cells undergoing mitosis, making pH3 a marker for labeling proliferating cells. In this manuscript, we described two immunostaining methods (immunofluorescence and immunohistochemistry) for detecting the pH3-positive cardiomyocytes in formalin-fixed, paraffin-embedded heart tissues.