Hello experts,
I want to know the reason why i am not getting nuclei staining on second staining(after done staining+destaining) with DAPI. The reagents i used for destaining are SDS-Glysine buffer(pH 2.0) at 50C/160 rpm

You haven’t provided us with some details, e.g., sample (sections (thickness?), cells), concentration and age of DAPI, incubation time, fluorescence filters, etc. Irrespective, DAPI staining is usually best at around neutral pH so one thing you could do is equilibrate your sample in a phosphate-buffered saline, pH7.0 (note that not all PBS buffers are the same!) for 5-10min or so, perhaps with 1-2 changes of PBS, prior to DAPI staining (and destain in the same PBS-like buffer; some samples require very little, if any destaining). I have no experience in destaining any DAPI-stained sample in the buffer and temp you specify, and suspect that this may be more related to staining/destaining nucleic acids in gels (e.g., http://nar.oxfordjournals.org/content/6 … abstract). In any event note that most (but not all) people would use different dyes to stain nucelic acids in gels, many with less potential toxicity compared with DAPI (e.g., see the ThermoFisher Scientific ‘The Molecular Probes Handbook’ Section 8.4). Hope this helps!

Last edited by Steve Lolait (12th Mar 2016 14:58:15)