A simple method for quantitating confocal fluorescent images

被引:299
作者
Shihan, Mahbubul H. [1 ]
Novo, Samuel G. [1 ]
Le Marchand, Sylvain J. [2 ]
Wang, Yan [1 ]
Duncan, Melinda K. [1 ]
机构
[1] Univ Delaware, Dept Biol Sci, Newark, DE 19716 USA
[2] Univ Delaware, Bioimaging Ctr, Delaware Biotechnol Inst, Newark, DE 19713 USA
基金
美国国家卫生研究院;
关键词
ImageJ; Confocal microscopy; Immunofluorescence; Mean fluorescence intensity (MFI); Cell counting; Protein quantitation; ROLES;
D O I
10.1016/j.bbrep.2021.100916
中图分类号
Q5 [生物化学]; Q7 [分子生物学];
学科分类号
071010 ; 081704 ;
摘要
Western blotting (WB), enzyme-linked immunosorbent assay (ELISA) and flow cytometry (FC) have long been used to assess and quantitate relative protein expression in cultured cells and tissue samples. However, WB and ELISA have limited ability to meaningfully quantitate relative protein levels in tissues with complex cell composition, while tissue dissociation followed by FC is not feasible when tissue is limiting and/or cells difficult to isolate. While protein detection in tissue using immunofluorescent (IF) probes has traditionally been considered a qualitative technique, advances in probe stability and confocal imaging allow IF data to be easily quantitated, although reproducible quantitation of relative protein expression requires careful attention to appropriate controls, experiment design, and data collection. Here we describe the methods used to quantify the data presented in Shihan et al. Matrix Biology, 2020 which lays out a workflow where IF data collected on a confocal microscope can be used to quantitate the relative levels of a molecule of interest by measuring mean fluorescent intensity across a region of interest, cell number, and the percentage of cells in a sample "positive" for staining with the fluorescent probe of interest. Overall, this manuscript discusses considerations for collecting quantifiable fluorescent images on a confocal microscope and provides explicit methods for quantitating IF data using FIJI-ImageJ.
引用
收藏
页数:21
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