Lastly, immune cells were labeled using a fluorescently conjugated anti-CD45 antibody. Nuclei of apoptotic cells were detected by terminal deoxynucleotidyl transferase dUTP nick end labeling (TUNEL) assay. CM boundaries were labeled with Wheat Germ Agglutinin (WGA) for the assessment of hypertrophy and capillaries were labeled with Isolectin B4 (IB4). CFs and their progeny were irreversibly tagged with tdTomato (TdT) using transgenic mouse models (genetic lineage-tracing), and mice were injected with 5-ethynyl-2′-deoxyuridine (EdU) 1 day before tissue collection to identify proliferating CFs. The fluorescently conjugated markers used in the study are detailed in Table 1. Details of tissue sectioning, staining and confocal microscopy are described in Janbandhu et al. ![]() The acquisition of high-quality 2D confocal images is necessary for accurate use of this pipeline, and is, as such, assumed. There are 4 CellProfiler pipelines and image sets used here for the analysis of: 1) cardiomyocyte (CMs) hypertrophy and vascularization 2) inflammation 3) cardiac fibroblast (CF) proliferation and 4) apoptosis. It is feasible that, using high-performance computing, tens of thousands of images could be analyzed for high throughput bioimage analysis, by calling these pipelines with the command line. Theoretically, there is no limit to the numbers of images that can be analyzed using this approach, the limiting factor here is computational power. No programming knowledge is necessary to run Fiji Macros or CellProfiler pipelines ( key resources table), so this protocol is highly accessible. ![]() These two open-source software are highly complementary when used in tandem for image-processing and segmentation, as well as for many other bioimage analysis needs ( Dobson et al., 2021). We outline an image analysis protocol for the automated analysis of post-ischemic cardiac parameters, using freely available software, Fiji ( Schindelin et al., 2012) and CellProfiler ( Carpenter et al., 2006 McQuin et al., 2018) ( key resources table).
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