We examined five nucleic acid binding fluorescent dyes, propidium iodide, SYBR Green I, YO-PRO-1, TOTO-3, and TO-PRO-3, for nuclear DNA staining, visualized by fluorescence and laser confocal microscopy. The optimal concentration, co-staining of RNA, and bleaching speeds were examined. SYBR Green I and TO-PRO-3 almost preferentially stained the nuclear DNA, and the other dyes co-stained the cytoplasmic RNA. RNAse treatment completely prevented the cytoplasmic RNA staining. In conventional fluorescence microscopy, these dyes can be used in combination with fluorescence-labeled antibodies. Among the dyes tested, TOTO-3 and TO-PRO-3 stained the DNAs with far-red fluorescence under red excitation. Under Kr/Ar-laser illumination, TOTO-3 and TO-PRO-3 were best suited as the nuclear staining dyes in the specimens immunolabeled with fluorescein and rhodamine (or Texas red).

In vivo electro-transfection efficiency and manner of transferred gene expression were investigated by fluorescence microscopic image analysis. Green fluorescent protein (GFP) gene was used as the genetic marker. Electroporation was carried out on the liver of live rats by use of disk electrodes mounted in the tips of tweezers, which were directly pressed onto the surface of a liver lobe in situ. Electroporation with eight electric pulses of 50 ms in duration at 50 V gave a good efficiency of transfection as judged by the induced GFP expression. Bright fluorescence of GFP appeared as dots, which were scattered around the area damaged by electroporation. The transfection efficiency increased as the amount of injected DNA was increased. The results indicate that the amount of induced gene expression can be controlled. Estimation of the efficiency of electro-gene transfer using the fluorescence of GFP and digital analysis of microscopic images was useful to determine the optimum conditions for local gene therapy in tissues and organs.z 1998 Federation of European Biochemical Societies.

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Together with our previously reported data, the above findings indicate that K(ATP) channels comprising SUR1 and Kir6.2 occur not only in beta cells but also in the alpha, delta, and pancreatic polypeptide cells of the pancreatic islets, suggesting that therapeutic sulphonylureas could act on these cells directly. [Diabetologia (1999) 42: 1204-1211]