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In reply to Steffen and Irina,

Steffen stated that Drosophila nuclei did not stain with PI until after RNAase treatment:
Propidium Iodide only stains permeabilised cells. Live cells will not stain. Mammalian cells and malaria parasites stain very well after fixation and so do many other cell types - although I have no personal experience with Drosophila. Lack of permeabilisation of some cell types may result in lack of or diminished staining. The fact that staining occurred after further treatment (RNAase treatment) suggests that there was a permeabilisation problem.

Irina stated that "PI stains denatured ssDNA  better than dsDNA, therefore undenatured nuclei should be stained MUCH longer....".
My understanding of the binding of PI to DNA is that it binds to dsDNA and dsRNA (by intercalation between the stacked base pairs) much more strongly than ssDNA or ssRNA. The binding within the cytoplasm, so I've been told, is mainly due to the PI binding to the dsRNA present in the ribosome complex. The DNA in fixed and permeabilised cells is not "denatured, i.e. single stranded". However, surrounding proteins certainly are "denatured" by the fixation process, allowing the PI more ready access to the DNA. Condensed chromosomal DNA stains very well with PI, as long as the PI can get in. In my experience, non-condensed nuclear DNA may not stain as well even after taking care to permeabilise the cells. This can be more noticeable when mounting in glycerol (which results in a spectral shift in the fluorescence emission). The fluorescence is more suitable for the typical dual label filter sets used in fluorescence microscopy if the mounting is done in buffer alone (for example PBS). A number of common anti-fade reagents also change the emission spectra of PI.

There are a number of better DNA stains now available, but PI is cheap and easy to use and so is certainly worth trying first.

Hope this is of some help, Alan.

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