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Fig. 4 Cryolesion results in loss of cardiomyocyte and endocardium-specific marker gene expression and in induction of apoptosis.
(A) In situ hybridization shows loss of cmlc2 (myl7) expression in the lesioned area at 3 and 7 dpi. The lesioned area is indicated by red arrowheads. Note the protrusions of cmlc2 positive areas (black arrow) extending into the lesion at 7 dpi. Scale bars are 100 μm. n = 3 hearts (9 sections each) for each uninjured, 3 dpi and 7 dpi. (B) Loss of cmlc2 expression is already evident in cryoinjured hearts at 1 dpi of cmlc2:GFP transgenic fish stained for GFP by immunofluorescence. Nuclei are stained with Dapi. Scale bar in overview is 100 μm, 25 μm in close-up. (C) Endocardial GFP expression is lost in cryolesions of fli1:eGFP transgenic fish at 1 dpi. (D) TUNEL assay on hearts at 1 dpi detects an increased number of apoptotic cells (white arrowheads) compared to uninjured hearts. Wound area is indicated by white dashed line and the periphery of the heart is marked by yellow dashed lines. Nuclei are stained with Dapi. Scale bar in overview is 100 μm. Scale bar in close up is 10 μm. n = 3 hearts each uninjured and 1 dpi.