However, the generation of reproducible data from fluorescent images relies on high-quality molecular probes/antibodies, optimized immunostaining protocols, confocal microscope settings that collect images within the linear range of the detectors, and choice of image analysis method appropriate to answer the scientific question of interest. Further, image analysis is particularly helpful when the tissue samples are limiting due to their small size or rarity, as more independent experiments are possible on tissue sections compared to traditional protein quantitation methods such as western blotting or flow cytometry which require much more starting material. These investigations have revealed counterintuitive phenomena and have led to new research directions as these approaches reveal the distribution of molecules in individual cells, instead of as an “average” concentration across a cell population as is obtained with methods such as western blotting or ELISA. In recent years, image analysis has been more frequently applied to quantitative measurement of numerous parameters including protein expression levels in cells and tissues, the number of cell nuclei in a tissue sample or section, tissue area and perimeter, protein co-localization with other proteins or cellular structures, concentration, and densitometry analysis, ,, , ].

Image analysis is playing an increasingly important role in scientific research as the computational power and digital camera technology necessary to collect and analyze images at high resolution have become more generally available. 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. 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. Here we describe the methods used to quantify the data presented in Shihan et al. 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. 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. 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.

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