A simple method for quantitating confocal fluorescent images
| Autor: | Yan Wang, Sylvain J. Le Marchand, Melinda K. Duncan, Samuel G Novo, Mahbubul H. Shihan |
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| Jazyk: | angličtina |
| Rok vydání: | 2021 |
| Předmět: |
0301 basic medicine
Mean fluorescence intensity (MFI) Microscope Confocal Quantitative proteomics Immunofluorescence Biophysics Biochemistry Flow cytometry law.invention Protein quantitation lcsh:Biochemistry 03 medical and health sciences 0302 clinical medicine law Confocal microscopy medicine lcsh:QD415-436 lcsh:QH301-705.5 Cell counting medicine.diagnostic_test Chemistry Fluorescence ImageJ Staining 030104 developmental biology lcsh:Biology (General) 030220 oncology & carcinogenesis Biomedical engineering Research Article |
| Zdroj: | Biochemistry and Biophysics Reports, Vol 25, Iss, Pp 100916-(2021) Biochemistry and Biophysics Reports |
| ISSN: | 2405-5808 |
| Popis: | 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. Graphical abstract Image 1 Highlights These simple methods:•Allow quantitation of molecules in small tissues using immunofluorescent (IF) detectionon tissue sections.•Generate data that correlate well with that obtained from other methods.•Yield reproducible data that expands the conclusions possible from IF imaging of tissues with complex cellular compositions. |
| Databáze: | OpenAIRE |
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