Molecular and Cellular Imaging Core

分子和细胞成像和组织学核心(核心C)将为以下每个人提供三项主要服务 1)成年小鼠心肌细胞的分离和功能分析 (2)腔静脉和心肌组织切片的显微镜分析，以及3)腔静脉和心肌组织细胞死亡的评估。 心肌样本。 1)在Core C中进行VCs的酶分离和电场刺激培养，以提供 在每个项目的特定条件下，用于分析VC功能的可靠且可重复的VC。 功能分析包括评估体外收缩能力和细胞内钙瞬变 分别使用数字化视频边缘检测系统和FURA2检测。 2)将提供一些基于显微镜的分析，以提供详细的免疫组织学和 对每个项目特定的VC和心肌组织切片进行荧光评估。这些分析 包括Vc和心肌组织切片的免疫组织学和形态分析、荧光 VCs和心肌组织切片分析及荧光/Forster共振能量转移(FRET) 风险投资中第二代信使和蛋白质相互作用的分析。 3)检测VC和心肌组织中细胞死亡情况。核心C使用分析来检测这两种细胞凋亡 和坏死性细胞死亡。每个项目都将获得末端脱氧核苷酸转移酶的分析 介导的dUTP缺口末端标记、caspase-3活性和Annexin V染色分离的VC和/或 用于评估细胞凋亡的心肌切片/提取物，以及乙锭高二聚体III(坏死)和 Hoechst(活细胞)染色Vc和肌球蛋白重链染色作为血管内皮细胞坏死的指标 心肌组织切片。 这一核心将继续被所有四个项目用来提供可重复使用的功能分析 分离的VC，以及VC和心肌组织的高质量显微镜和细胞死亡分析。

The molecular and cellular imaging and histology core (Core C) will provide three main services to each of the three projects described: 1) isolation and functional analysis of murine adult ventricular cardiomyocytes (VC), 2) microscopy analysis of VC and myocardial tissue sections, and 3) evaluation of cell death in VC and myocardial samples. 1) Enzymatic isolation and electrical-field stimulated culture of VCs will be performed in Core C to provide reliable and reproducibly viable VC for analysis of VC function under conditions specific to each project. Functional analyses include assessment of in vitro contractility and intracellular calcium (Ca2+) transients using a digitalized video-edge detection system and FURA2 detection, respectively. 2) A number of microscopy-based assays will be available to provide detailed immunohistological and fluorescence assessment of VC and myocardial tissue sections specific to each project. These analyses include immunohistological and morphological analyses of VC and myocardial tissue sections, fluorescence analysis of VCs and myocardial tissue sections and fluorescence/forster resonance energy transfer (FRET) analysis of 2nd messenger generation and protein interactions in VCs. 3) To evaluate cellular death in VC and myocardial samples. Core C uses assays to detect both apoptotic and necrotic cell death. Each project will have access to analyses of terminal deoxynucleotidyl-transferase mediated dUTP nick end (TUNEL) labeling, caspase-3 activity and Annexin V staining isolated VC and/or myocardial sections/extracts for evaluation of apoptosis, as well as ethidium homodimer III (necrosis) and Hoechst (living cells) staining of VC and myosin heavy chain staining as an indicator or necrosis in myocardial tissue sections. This core will continue to be utilized by all four projects to provide reproducible functional analyses of isolated VC, as well as high quality microscopy and cell death analyses of VC and myocardial tissue.