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Dear fellow researchers,

I have been facing some problem with staining nucleus with DAPI. I use the standard protocol for fixation 4%PFA+0.1% TX-100+2%BSA.

I use 0.2ug/ml conc. of DAPI for 5 minutes. Before mounting my cover slides, the nuclear staining is very specific and no artefacts were visible. However, after mounting (with Sigma-Aldrich mounting medium), I have started observing artefacts. Images are attached for your reference. 

I usually keep my samples at room temperature for an hour to let the mounting medium settle down and then move samples to 4 degrees overnight and observe them the next day. I do not think that temperature here is an issue because the other marker did not show any problems.

Another issue is that the artefacts of nuclear staining are not ubiquitous. Even after mounting, some cells stained nucleus fine but others showed cytoplasmic or membrane staining. 

Is there any reason that you guys can think of? Or if you have encountered similar situation before? Thanks in advance. 

Why is DAPI giving artefacts with nuclear staining? - ResearchGate. Available from: https://www.researchgate.net/post/Why_is_DAPI_giving_artefacts_with_nuclear_staining [accessed Sep 18, 2016].

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  • $\begingroup$ Hi Udesh. Welcome to Biology.SE. Can you post the images here? It would be easier for people to check them here instead of visiting other websites. Also, can you please mention what cells were these? Did you try gentle washing to remove excess DAPI? $\endgroup$ – WYSIWYG Sep 18 '16 at 9:35
  • $\begingroup$ Hello! Unfortunately I could not attach the images here. But, in my post there is a link to the same post on research gate. Could you take a look at it? It has images attached there. Yes, I washed three times but it did not help. These were MDA-MB-231 breast cancer cells. $\endgroup$ – Udesh Dhawan Sep 19 '16 at 12:09

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