Hi,
I am trying to co-label tissue sections with Brdu and To-pro-3. Interestingly when I immunostain for Brdu and Neun/GFAP I can see very nice staining but when I include Topro-3 no Brdu positive signal is seen. Why Brdu signal vanish when TO-Pro3 (nuclear stain) is included? I have tried both 2N HCl treatment and heat mediated antigen retrieval before immunostaining for Brdu. In both cases for some reason To-Pro3 signal is preventing the Brdu signal. Can anyone tell me what is the reason?
Probably the To-pro-3 overshadows the Brdu staining as it has a high labeling density for DNA. And as it intercalates in between the bases it might also change the binding behavior and the interactions between the Brdu and its antibody. Maybe it is worth to try DRAQ5 as nuclear staining?
You can try to use DAPI or fix for 5 min with pfa 4% after brdu stainning, wash with pbs and then add the to-pro-3.
@ Matthias, Thank you for the reply. I purchased DRAQ5 tried staining one section for 5 mins (1:1000) as recommended by the vendor (Sigma). I see lot of background which was not the case with Topro-3. Any tips to improve the staining?
@ Bruna, Thank you for the reply. I can't try DAPI as we don't have the UV laser to excite it. I can try fixing the cells before Topro-3 addition. I will update you. Thanks!
To decrease background you could try to extend the washing step after the DRAQ5 (we do 2x 5 min for 8 um sections) and rinse in aq dest afterwards. Which solution do you use to dilute the DRAQ5? What kind of microscope and laser lines do you use? With an SP8 you can narrow the PMT range so you can eliminate the background quite effectively.
Matthias, I use TBS for diluting DRAQ5. Currently I am doing one staining, I will try rinsing the sections more. I use confocal (Leica SP2) red and far red lasers.
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