What should I consider when choosing counterstain?

The counterstain one chooses is dependent on the color of the end product or fluorochrome. DAPI is the most common fluorescent nuclear counterstain. It is also important to choose a counterstain that does not cause any spectral overlap and bleedthrough. For example, DAPI should not be used in conjunction with Alexa Fluor 405, dyLightTM 405, or HiLightTM 405 because they all emit blue fluorescence. The counterstain of choice should be of a different color to the detection system. This is so one can differentiate the signals from the secondary antibody and counterstain. Nuclear counterstains should not be too strong during the detection of nuclear antigens, otherwise it could mask positive staining. Haematoxylin and methyl green are the most commonly used nuclear counterstains. For cytoplasmic staining, eosin is typically used together with hematoxylin.