Hi Victoria,

the easiest way to prevent his problem is to do a sequential labeling; first the copper mediated click reaction with the Azide 488, wash with a buffer containing EDTA to remove the copper and continue with the immunolabeling with the streptavidin 568.

Since the click reaction is irreversible there is no risk of losing the 488 signal with the added washing/incubation steps.

I hope this helps, best regards,

Rob

Would EDTA in PBS by acceptable? Also what concentration of EDTA should be used? 

Hi Victoria,

In my experience 5mM EDTA is sufficient to chelate the ammount of copper normally used in click reactions (I've used it as an addition to a click reaction to make a negative control). As a buffer PBS should be ok,  just to keep things easy I would recommend using the same buffer you plan to use in the immunolabeling.

Best regards,

Rob

Thanks for the suggestion. We tried a 5mM EDTA wash for 5 minutes and it did not seem to fix the problem. We are following the protocol below. We also tried replacing the copper solution with Cu-TBTA complex from lumiprobe. When we do that we get no EdU labeling and the tissue looks very bright even when the copper is used at low concentrations and shows no EdU labeling. 

Permeabilize 10 min in PBS + 0.1% Triton X-100

Wash 3x with PBS

Prepare label mix

Incubate cells 30 min. with label mix 

Wash cells 3x 5 minutes with PBS

Then label with other antibodies

Stock solutions:

Sulfo-Cy3-Azide: 4 mM in H2O (3 mg in 1 ml H2O) (final 8 µM)

CuSO4.5H2O: 200 mM in H2O (250 mg in 5 ml) (final 2 mM)

Ascorbic acid 200 mg/ml 

Working solution:

PBS 888 µl

CuSO4 10 µl

Azide-dye 2 µl

Ascorbate 100 µl

Since washing with EDTA doesn't seem to have any effect, I doubt that the loss of signal is due to the copper.

It could be that the ascorbic acid used in the click reaction damages the epitopes you target in the immunolabeling. When I did Click labeling on thawed cryo-sections for electron microscopy I found out that ascorbic acid damaged the ultrastructure conciderably, I switched to using 100uM TBTA in the click reaction which worked ok for me.

Since you already indicate that the commercial Cu-TBTA complex doesn't work you could consider lowering the ascorbic acid concentration to 50mM (the lower level reported by Salic et al. 2008, doi: 10.1073/pnas.0712168105) or use a combination of 1mM ascorbic acid and 100uM TBTA (in which the ascorbic acid converts Cu(II) to Cu(I) and the TBTA stabilizes the Cu(I).

Another option is to first do  the immuno-labeling,  fix for 15 minutes with 1% PFA in PBS, wash 3x 5 minutes with PBS and continue with the click reaction.

I hope this helps,

best regards,

Rob

Hi thanks for responding again. We tried the Cu-TBTA at 100uM and the ascorbic acid at 1mM. However we still get no EdU labeling in the tissue and the background is very high. It is also possible the background is masking the EdU labeling. Do you know what may be causing the background? Should the azide concentration be changed when using the Cu-TBTA? When we used the copper sulfate the background was very low.