Fluorochrome DAPI

A UV laser is needed for exciting the blue-fluorescing agents, 4, 6-diamidino-2-phenyhndole (DAPI) and Hoechst 33342, which are DNA-intercalating stains, and for indo-1, a fluorescent calcium chelator dye. Violet diode lasers that are offered in some newer instruments accommodate fluorochromes such as Cascade Blue, Pacific Blue, and cyan fluorescent protein, and are also capable of exciting DAPI (Shapiro and Perlmutter 2001 Telford et al., 2003). 

There are other fluorochromes that can be used for DNA analysis. The following three are the most straightforward and reproducible with regard to application mithramycin, 4, 6-diamidino-2-phenylindole hydrochloride (DAPI), and Hoechst 33258. Concentrations must be determined empirically (see Chapter 30). 

These methods use fluorescent labels, such as propidium iodide, ethidium bromide, or DAPI (4, 6 -diamidino-2-phenylindole), which are incorporated into the DNA, allowing chromatin condensation and nuclear fragmentation to be visualized under a microscope with the appropriate fluorescence filters. To allow fluorochromes to enter the cells and reach the nucleus, the cells need to be prepermeabilized, for example, with 70% ethanol at -20°C. LMW-DNA fragments may be lost by the permeabilization, decreasing the amount of DNA inside the cells. The lower nucleic acid concentration results in a lower fluorescence intensity in apoptotic cells, which can be detected by fluorescence microscopy or flow cytometry (Calle et al., 2001). 

The more sensitive and convenient method of PolyP detection in situ is fluorescence microscopy using fluorochromes of the type 4/,6/-diamino-2-phenylindole.2HCl (DAPI), which is commonly used for DNA detection. At a high concentration (50 mg ml 1), it also stains PolyP granules and lipid inclusions (Allan and Miller, 1980 Tijssen et al, 1982 Streichan et al, 1990). DAPI-DNA fluorescence is blue-white, while DAPI-PolyP and DAPI-lipid fluorescence is yellow. The lipid fluorescence is weak and fades in a few seconds, while the PolyP granules appear bright yellow, thus allowing discrimination of the above types of cell inclusions (Streichan et al, 1990). 

DAPI is the fluorochrome of choice when dealing with small intraspedfic differences in maize. DAPI binds to both A-T and G-C base pairs and fluoresces when bound to either type. However, the fluorescence when DAPI is bound to A-T-rich DNA is greater than when bound to G-C-rich DNA. Comparisons of DAPI-stained nuclei to Feulgen-stained nuclei have demonstrated that this base pair specificity does not preclude the use of DAPI in detecting small DNA differences in maize.5... 

After the numbers of nuclei have been determined, the fluorochrome is added to the nuclei. A staining concentration of 2.5 pig of DAPI per 106 nuclei (determined by a titration curve) is used. The stock solution of DAPI consists of 1 pig of DAPI per 1 pi of distilled water. Critical to proper staining is the accurate determination of the number of nuclei to be stained (fluorochrome nuclei ratio). This caveat should be heeded with all fluorochromes. The titration curve of DAPI is such that significant over- as well as underestimates of the number of nuclei result in distortions of the resulting data. Similar results, although not so extreme, have been observed with other fluorochromes. After staining, the samples are kept on ice until examined. 

Incubate for 10 min with 100 /i.1 normal goat serum, followed by a 30-min incubation with 50 fluorochrome-conjugated secondary antibody in PBT. Wash two times for 3 min with 100 /i.1 PBT to remove excess antibody. If necessary, nuclei can be counterstained with a solution of 5 ju,g/ml DAPI (Sigma cat. D9542) in PBT for 5 min. 

Primary antibodies. See Tables 2 and 3. Fluorochrome-conjugated secondary antibodies. See Note 3. 4, 6-Diamidino-2-phenylindole (DAPI). 

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