High-resolution confocal imaging is a vital tool for analyzing the 3D architecture and detailed spatial distribution of cells in situ. However, imaging of skeletal tissue has remained technically challenging because of its calcified nature. Here we describe a protocol that allows high-resolution imaging of skeletal tissue with preservation of cellular morphology and tissue architecture. The procedure involves tissue fixation, decalcification and cryosectioning of the mouse skeletal tissue to generate thick sections. The thick sections generated by this procedure are not only compatible with the analysis of genetically expressed fluorescent proteins but they also preserve antigenicity, thus enabling diverse combinations of antibody labeling. Further, this procedure also permits other fluorescence techniques such as TUNEL and ethynyl deoxyuridine (EdU) incorporation assays. Images resulting from the confocal imaging can be assessed qualitatively and quantitatively to analyze various parameters such as distribution and interrelationships of cell types. The technique is straightforward and robust, highly reproducible and can be completed in ∼11 d.

This study suggests that in BPH-1 cells the transcription factor SNAI2/Slug is important for EMT initiation, while the ZEB family of transcription factors in cooperation with the miR-200 family may oppose the reversal of the EMT phenotype.

The hyperlipidemic mouse model HypoE/SRBI −/− has been shown to develop occlusive coronary atherosclerosis followed by myocardial infarctions and premature deaths in response to high-fat, highcholesterol diet (HFC). However, the causal connection between myocardial infarctions and atherosclerotic plaque rupture events in the coronary arteries has not been investigated so far. The objective of this study was to assess whether diet-induced coronary plaque ruptures trigger atherothrombotic occlusions, resulting in myocardial infarctions in HFC-fed HypoE/SRBI −/− mice. Methods: HypoE/SRBI −/− mice were characterized with respect to the individual dynamics of myocardial infarctions and features of infarct-related coronary atherosclerosis by serial noninvasive molecular and functional imaging, histopathology, and a pharmaceutical intervention. Detailed histologic analysis of whole mouse hearts was performed when spontaneously occurring acute myocardial infarctions were diagnosed by imaging. Results: Using the imaging-triggered approach, we discovered thrombi in 32 (10.8%) of all 296 atherosclerotic coronary plaques in 14 HFC-fed HypoE/SRBI −/− mice. These thrombi typically were found in arteries presenting with inflammatory plaque phenotypes. Acetylsalicylic acid treatment did not attenuate the development of atherosclerotic coronary plaques but profoundly reduced the incidence of premature deaths, the number of thrombi (7 in 249 plaques), and also the degree of inflammation in the culprit lesions. Conclusion: HFC-induced ruptures of coronary plaques trigger atherothrombosis, vessel occlusions, myocardial infarctions, and sudden death in these mice. Thus, the HypoE/SRBI −/− mouse model mimics major features of human coronary heart disease and might therefore be a valuable model for the investigation of molecular and cellular parameters driving plaque rupture-related events and the development of new interventional approaches.

The plasticity of differentiated adult cells could have a great therapeutic potential, but at the same time, it is characteristic of progression of serious pathological states such as cancer and fibrosis. In this study, we report on the application of a real-time noninvasive system for dynamic monitoring of cellular plasticity. Analysis of the cell impedance profile recorded as cell index using a real-time cell analyzer revealed its significant increase after the treatment of prostate epithelial cells with the transforming growth factor-β1. Changes in the cell index profile were paralleled with cytoskeleton rebuilding and induction of epithelial–mesenchymal transition and negatively correlated with cell proliferation. This novel application of such approach demonstrated a great potential of the impedance-based system for noninvasive and real-time monitoring of cellular fate.