Publication protocol
The following methods were utilized: immunohistochemical staining with various combinations of antibodies of frozen cardiac sections from prenatal stages: 12.5, 13, 14, 15, 16, 17, 18 dpc and from postnatal days 1, 4, 8, 9, 14, 21, and adult animals (4–8 weeks). Molecular marker‐phenotype of mouse cardiac lymphatic plexus was determined by the use of criteria on the presence or absence of the selected antigens among the following: Lyve‐1 (rat, R&D Systems, #MAB2125; rabbit, Angiobio, #11034), Prox‐1 (rabbit, Abcam, #ab37128), VEGFR2 (Flk‐1, goat, R&D Systems, #AF644), CD31 (rat, BD Biosciences, Pharmingen, # 550274; goat, Santa Cruz Biotechnology Inc., #sc 1506), VEGFR3 (rabbit, MyBioSource, # MBS625970; rat, BD Pharmingen, #552857), CD144 (rat, BD Biosciences Pharmingen #550548), vWF (von Willebrand factor, sheep, Abcam, #ab11713), CD34 (rabbit, Genway Biotech, Inc. #GWB‐BBp214), CD133 (rat, eBioscience, #14–1331). Separate sets of immunostainings were performed with various combinations of the primary antibodies mentioned above (as presented in Table 1). Whole‐mount immunohistochemical staining of prenatal hearts at stages 12, 12.5, 13, 14, 15, 16, 17, 18 dpc with anti‐Lyve‐1, anti‐Prox‐1, or anti‐VEGFR3 antibodies. On average, three hearts from each stage of development were used for each staining method (either whole‐mount or tissue section immunohistochemistry). The secondary antibodies were: Fluorescein (FITC)‐conjugated donkey anti‐sheep IgG‐FITC (Jackson, #713‐095‐147), Cy™3‐conjugated donkey anti‐rabbit IgG, (Jackson #711‐165‐152), AlexaFluor® 647‐conjugated donkey anti‐rat IgG, (Jackson #712‐605‐153), Fluorescein (FITC)‐conjugated donkey anti‐Goat IgG (Jackson #705‐095‐147). Sections were visualized and analyzed under a confocal microscope (Leica), while whole‐mount‐stained hearts, and India ink‐injected hearts under a stereoscope (Nikon).
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