I'm prepping drosophila ovaries for immunofluorescence. The protocol I'm using uses RNAse A and Propidium iodide for the nuclear stain. My understanding is that if I use DAPI I don't need a RNAse as DAPI doesn't stain RNA. Do I need to worry about my antibodies possibly attaching to RNA or is there another reason why RNAse should be used in this case?
No, you only need RNAse treatment if you use PI because it binds to RNA as well. DAPI binds only to DNA.
Hello Jillian Porter
RNase treatment is not required when using DAPI. DAPI is fairly specific to DNA. But for your information DAPI does bind to RNA but with lower affinity and with less intense fluorescence. When bound to RNA evidently through AU-selective intercalation, the emission is about 5-fold weaker, and the excitation maximum is shifted into the green range.
Best.
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