I performed fluorescence stainings on brain formalin-fixed paraffin embedded samples (zebrafish). I stained TH+ neurons and counterstained with DAPI.
In tissues coming from 2 specific animals, DAPI (diluted 1:1000) is not working. For all the other samples, belonging to other animals, DAPI is working perfectly.
I am positive that this outcome is not user-dependent, because in the same session I stained many slides, and in some of them the DAPI worked, in some others it didn't. TH staining worked perfectly for all tissues.
P.S. I performed an antigen retrieval with citrate buffer pH 6.0 for 15 minutes at 95°C in a water bath.
Please help! I don't know what could have happened!
Hi,
have you tried 5 µg/ml DAPI in 0.5−1% (v/v) Triton X-100/1× PBS solution or to use Hoechst instead of DAPI?
Best, Anne
Hi Jacopo Grisotto
Are you using Hoechst? Do you stain with Hoechst at last before mounting?
I would recommend you to use DAPI with mounting medium (ibidi or vectashield).
Thanks
I dissolved the DAPI in PBS, without triton. But triton was present (0.3%) in the washes that preceeded the incubation with DAPI. Would you recommend to dissolve the DAPI in PBS added with triton?
Samir Ranjan Panda I am only using DAPI, I've never used Hoechst. Would you recommend it?
Do you mean to dissolve DAPI into the mounting medium?
Thank you all anyway!
Hi Jacopo Grisotto,
you can by Vectashield w/ DAPI directly. I used it also in the past.
Hoechst can pass the membrane also without any permabilisation. Just in case you have any doubt that this would be an issue.
Good luck!
Best, Anne
For zebrafish brain sections I have used DAPI at a final concentration of 300 nM (in PBS) for two minutes, followed by 3 washes. It worked perfectly fine.
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