Stem Cell Institute, Paul and Sheila Wellstone Muscular Dystrophy Center, and Department of Neurology, University of Minnesota Medical School, Minneapolis, Minnesota, USA.
J Histochem Cytochem. 2011 Jan;59(1):60-7. doi: 10.1369/jhc.2010.956730.
Staining for β-galactosidase activity for whole tissues, sections, and cells is a common method to detect expression of β-galactosidase reporter transgene as well as senescence-dependent β-galactosidase activity. Choice of fixatives is a critical step for detection of β-galactosidase activity, subsequent immunostaining, and enzymatic digestion of tissue to dissociate cells. In this report, the authors examined several aldehyde and alcohol fixatives in mouse skeletal muscle tissues for their efficiency at improving detection of β-galactosidase activity as well as detection by immunostaining. In addition, fixatives were also analyzed for their efficiency for collagenase digestion to isolate single muscle fibers on postfixed β-galactosidase-stained whole skeletal muscle tissues. The results show that fixing cells with isopropanol yields the greatest reliability and intensity in both β-galactosidase staining as well as double staining for β-galactosidase activity and antibodies. In addition, isopropanol and ethanol, but not glutaraldehyde or paraformaldehyde, allow for the isolation of single muscle fibers from the diaphragm and tibialis anterior muscles following postfixed β-galactosidase staining. Using this method, it is possible to identify the amount of cells that occupy the satellite cell compartment in single muscle fibers prepared from any muscle tissues, including tibialis anterior muscle and diaphragm.
对整个组织、切片和细胞进行β-半乳糖苷酶活性染色是检测β-半乳糖苷酶报告基因表达以及衰老相关β-半乳糖苷酶活性的常用方法。固定剂的选择是检测β-半乳糖苷酶活性、随后进行免疫染色以及组织消化以分离细胞的关键步骤。在本报告中，作者研究了几种醛类和醇类固定剂在小鼠骨骼肌组织中的效果，以提高β-半乳糖苷酶活性的检测效率以及免疫染色的检测效率。此外，还分析了固定剂在胶原酶消化以分离固定后β-半乳糖苷酶染色的整个骨骼肌组织中的单个肌纤维方面的效率。结果表明，异丙醇固定细胞在β-半乳糖苷酶染色以及β-半乳糖苷酶活性和抗体的双重染色方面具有最大的可靠性和强度。此外，异丙醇和乙醇可以分离固定后β-半乳糖苷酶染色的膈和比目鱼肌中的单个肌纤维，而戊二醛或多聚甲醛则不行。使用这种方法，可以从任何肌肉组织（包括比目鱼肌和膈肌）制备的单个肌纤维中鉴定占据卫星细胞隔室的细胞数量。
