Mozdziak P E, Fassel T, Gregory R, Schultz E, Greaser M L, Cassens R G
Department of Meat and Animal Science, University of Wisconsin, Madison 53706.
Biotech Histochem. 1994 Sep;69(5):249-52. doi: 10.3109/10520299409106296.
A nonisotopic, double fluorescence technique was developed to study myogenic satellite cell proliferation in posthatch turkey skeletal muscle. Labeled satellite cell nuclei were identified on enzymatically isolated myofiber segments using a mouse monoclonal antibody (anti-BrdU) followed by fluorescein-5-isothiocyanate (FITC) conjugated goat anti-mouse IgG secondary antibody. Myofiber nuclei (myonuclei+satellite cell nuclei) were counterstained with propidium iodide (PI). The myofiber segment length, myofiber segment diameter, and the number of PI and FITC labeled nuclei contained in each segment was determined using a Nikon fluorescence microscope, a SIT video camera and Image-1 software. Data collected by three different operators of the image analysis system revealed 5.0 +/- 1.4 satellite cell nuclei per 1000 myofiber nuclei and 5284 +/- 462 microns3 of cytoplasm surrounding each myofiber nucleus in the pectoralis thoracicus of 9-week-old tom turkeys. BrdU immunohistochemistry coupled with the new approach of PI staining of whole myofiber mounts is an effective combination to allow the use of an efficient semi-automated image analysis protocol.
开发了一种非同位素双荧光技术，用于研究孵化后火鸡骨骼肌中肌源性卫星细胞的增殖。使用小鼠单克隆抗体（抗BrdU），然后用异硫氰酸荧光素（FITC）偶联的山羊抗小鼠IgG二抗，在酶分离的肌纤维片段上鉴定标记的卫星细胞核。肌纤维核（肌核+卫星细胞核）用碘化丙啶（PI）复染。使用尼康荧光显微镜、SIT摄像机和Image-1软件确定每个片段中肌纤维片段长度、肌纤维片段直径以及PI和FITC标记核的数量。图像分析系统的三名不同操作人员收集的数据显示，9周龄雄性火鸡胸大肌中每1000个肌纤维核有5.0±1.4个卫星细胞核，每个肌纤维核周围有5284±462立方微米的细胞质。BrdU免疫组织化学与全肌纤维标本PI染色的新方法相结合，是一种有效的组合，可用于高效的半自动图像分析方案。
