TO-PRO-3 is an optimal fluorescent dye for nuclear counterstaining in dual-colour FISH on paraffin sections

Confocal laser scanning microscopy (CLSM) is an extensive but reliable tool for assessing the hybridisation signals in fluorescence in situ hybridisat ion (FISH). Most CLSMs are equipped with an argon-laser and a helium/neon-l aser illumination system with excitation wavelengths of 488, 543 and 633 nm . A protocol for an optimal nuclear counterstaining in combination with dua l-colour FISH for these laser illumination systems has not been established so far. Here, we determined the suitability of eleven dimeric and monomeri c cyanine nucleic acid stains on paraffin sections of breast carcinoma spec imens in combination with dual-colour FISH (Her-2/neu and centromere 17) fo r CLSM application. Strong staining of cell nuclei was observed for TO-PRO- 3 and YO-PRO-3, YOYO-1 and propidium iodide (PI), but only TO-PRO-3 showed specific staining of nuclei without any staining of the cytoplasm. A specif ic emission in exclusively one distinct fluorescence channel was shown for TO-PRO-3 (633 nm excitation) as well as YOYO-1, BO-PRO-1 and Sytox Green (4 88 nm excitation), evaluated by a CLSM and confirmed by 3-D fluorescence sp ectra. High stability of fluorescence intensity was shown for the far-red d yes TO-PRO-3, YO-PRO-3, YOYO-3 and Syto-59 as well as YOYO-1 and PI. Only T O-PRO-3 was due to its high specificity and stability suitable for detectio n of an amplification of the Her-2/neu gene by dual-colour FISH and CLSM ev aluation.