IMAGING TO MEASURE FORMATION OF PUNCTA IN THE CYTOPLASM
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 while the other leads to accumulation of the protein marker in cytoplasmic puncta or spots.
After creating a region of interest (ROI) that covers the cytoplasm, puncta within that 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.