Immunofluorescence_Destaining_Dapi_no staining after destain with SDS-Glycene pH-2
Hi Shaik,
Is it possible that your DAPI has been exposed too much to light?? With the exposure to light it loses its fluorescent properties.
Best wishes
Thanks for you reply Marc Antoni,
No, I did not expose it to light much time. On the first time( DAPI 5 mins incubation) staining there is very good fluorescence but the next time(5 mins incubation) no fluorescence. I think low pH(2.0) or SDS- Glycine buffer effected on nuclei.
Hi jordan,
I am not destaining with DAPI. I am performing staining and then destaining on the same slides. So 1st i do Staining of insulin then i destain it and later i do restaining again on same slide. i am only staining with DAPI but not in destaining step.
Dear Shaik,
I face the same problem. Did you solve the problem by now?
Best Mijke
Dear Mijke,
Yes i solved this problem most extent. So previously i was taking Dapi concentration 1:3000(Dapi:PBS). This will not work after 2nd or 3rd rounds of destaining. To get a clear signal use TBS and increase the concentration, you can increase the concentration respect to the rounds like 1:2000, 1:1500(Dapi:TBS). Because, after every round we loss some genetic material from sections. And i increased the thickness of tissue also from 4um to 8um according to my experiment. i hope this will help you
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