Figure 3.

The Apex-Resected Zone in C57Bl/6 Hearts Reveals Limited Vascularization and Numbers of Proliferating Cardiomyocytes

(A) Cryosectioned C57Bl/6 AR hearts (n = 4–8) were immunostained for aSMA, CD31, and NG2/CD31. Representative images were processed (contrast/brightness and color balance) equally in Photoshop to enable merging. An asterisk (^∗) indicates lesion area, whereas a number sign (#) refers to nondamaged myocardium.

(B) Quantitative real-time PCR for Cd31 of sham and AR hearts (n=4) at indicated time points. Quantitative real-time PCR raw data were normalized against the stably expressed B2M and β-actin genes (see Figure 2).

(C–F) Paraffin-embedded C57Bl/6 AR and sham hearts (n = 5) from EdU pulse-chase labeled animals were sectioned and immunostained for EdU/cardiac myosin/collagen I/DAPI to enable identification of proliferating cells in total and proliferating cardiomyocytes specifically. Images were taken in four different zones (white boxes in C and areas magnified in F) of each heart and used for cell number quantification (D and E) as described in Experimental Procedures.

(G) Quantitative real-time PCR for Meis1b of sham and AR hearts (n = 4) at indicated time points. Quantitative real-time PCR raw data were normalized against the stably expressed B2M and β-actin genes (see Figure 2).

(H) Meis1b is downregulated with cardiac development. Hearts were microdissected from C57Bl/6 mice at indicated time points and used for quantitative real-time PCR. Primers used were previously described by Mahmoud et al. (2013). Each biological experiment (n = 3–6) included between one and six hearts. qPCR raw data were normalized against Gapdh and β-actin, which were stably expressed as determined by the qBase+ platform (M:0.306, CV: 0.106) (Hellemans et al., 2007, Vandesompele et al., 2002). Statistical significance was tested using a one-way ANOVA with a Dunnett’s multiple comparison posttest. E11.5, embryonic day 11.5.