In reply to Steffen,

The images of testis tissue on your web site are certainly impressive. You certainly do have "black holes" where the nucleus should be when stained with PI. This still looks like a permeabilisation problem to me. The change in detergent (from 0.3% Tween to 1% Triton) could account for the difference after RNAase treatment. However, this tissue will have an unusually high level of PI staining in the cytoplasm as these cells are very active. The PI staining is mainly associated with the ribosome and so high levels of active translation will result in more cytoplasmic labelling. Even so I'd be surprised if the stain in the cytoplasm was greater than the nucleus. If you treat the tissue with 1% Triton without the RNAase treatment you'd probably end up with at least some labelling in the nucleus.

PI normally stains the DNA in the nucleus very much brighter than the RNA in the cytoplasm. Maybe other people have found your strange PI staining effect in other highly active tissue.

Good luck, Alan.

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