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.

The nuclear marker serves a few important purposes. Firstly, it acts as a reference for the automated microscope to focus. The Hoechst dye is very bright and resistant to photobleaching so has become an industry standard for this function. Secondly, the Hoechst signal is used to count nuclei in each image and subsequently each well. This simple measurement is often informative providing basic information regarding toxicity, proliferation, cell density and even cell cycle state for some cell types. Finally, the Hoechst marker is used as a spatial reference with which other dyes may be quantified. In the example shown, the region surrounding each nucleus is quantified for levels of the protein depicted in green.

Specific proteins are typically detected via indirect labelling with a primary antibody and a fluorescently-conjugated secondary antibody that binds the primary. RNA molecules can be detected using commercially-available kits enabling single copy detection and localization of custom target sequences (QuantiGene, ThermoScientific & SmartFlare, Millipore).

Using this approach it is possible to quantify 4-5 markers in a multiplexed fashion relative to the nuclear signal.

What if the subcellular localization of the marker changes?
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