In the simple schematic shown to the right, untreated cells express a moderate amount of the marker protein (depicted in green), which appears to be cytoplasmic. The nuclei (depicted in blue) are typically labeled using Hoechst 33342 (learn why we use Hoechst here).

In the previous example levels of the protein marker depicted in green changed but it remained cytoplasmic. In this schematic, one condition leads to an increase in cytoplasmic levels and formation of puncta coincident with the nuclear mask derived from the Hoechst image. The condition shown in the bottom right of the schematic depicts no change in intensity or localization of the protein marker but a change from diffuse to punctate distribution of signal within the cytoplasm.

After creating two regions of interest (ROIs), one that covers the nucleus and a second that corresponds to the cytoplasm, puncta within each ROI can be detected, counted and their properties such as size, shape and intensity calculated.

Using this approach, it is possible to quantify changes in protein expression level AND formation of puncta in multiple ROIs for 4-5 markers in a multiplexed fashion relative to the nuclear signal.

See what other types of analyses can be performed
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