I have marked my U87 glioma cells several times with a primary antibody made in rabbit and then revealed with Alexa 488 which marks a cytoplasmic protein and never had any problems with non-specific marking. However, since I have been using Alexa 594 with the same primary antibody, I am seeing strong nuclear marking that I did not have before.
The protocol I use is as follows:
Cell Fixation 4% formaldehyde 10 minutes,
Permeabilisation 0.05% Triton X100 5 minutes
Blocking incubation 10% FBS 2% NDS 1h
Incubation 4°C overnight with 1st Ab and the next day 2h RT with Donkey Anti Rabbit Alexa (488 or 594). Then 10 minutes incubation with DAPI.
Is it possible there is contamination in the Alexa 594 perhaps with a primary antibody that marks the nucleus?
Thank you!
Hi Daniele:
There are a few things with DAPI nuclear staining:
- The DAPI has a long emission tail which cross talks with other channels
- There is a possibility of UV photo conversion of DAPI to "undergo UV-induced photoconversion to form green-emitting fluorophores" which might be having emission properties in the detection range (https://biotium.com/support/tech-tips/tech-tip-avoiding-artifacts-from-uv-photoconversion-of-dapi-and-hoechst/)
- Imaging DAPI signal last might solve your problem or use some of the NIR nuclear dyes whose emission is red-shifted away from your other signals
Good luck!
@Sathya Srinivasan
Hello Daniele
Did you run a control only with secondary antibody (no primary) ? If not, please do.
Best Wishes.
I agree with Malcolm Nobre and to a degree, with Sathya Srinivasan while DAPI may degrade and leak into the other channels under the UV light Daniele Campisi report strong nuclear signals with degraded DAPI usually producing weak fluorescence. To summarize already suggested:
1. No primary control incubate the cells with only a secondary antibody of interest. 595 and see if they visualize the nuclei thus confirming the nonspecific binding.
2. No DAPI control perform the experiment without using DAPI.
3. You may consider the possibility that protein of interest is present in the nuclei due to some change in the cell culture behavior there are several ways to confirm it. Incubate the sample with both 488 and 595 secondaries against the same species as primary (i.e. primary rabbit host and 488 and 595 anti-rabbit secondary) and detect both channels. Only signals that colocalize and produce yellow signals in the merged images are true any signal that present in only one of the channels is non-specific. If this is indeed the nuclear presence of the target of interest it can be further confirmed by nuclear extraction and WB.
Dears all, thank you for your helpful advices!! Malcolm Nobre I did this and no signal was detected. Anton Lennikov and Sathya Srinivasan , if the problem had been caused by the long emission tail of the DAPI I would have mainly had the problem with the green (488) but instead I only had it with the alexa 594! However, if I use Alexa 488 for the same primary antibody, the nuclear marking is not present!
Lately I have tried to stain my endogenous protein with an Alexa 405 (without using DAPI of course) and I had a strong nuclear marking here too. I am ok with the Alexa 488 only and I do not understand why. If my secondary Abs (alexa 405 and 594) had been contaminated I would have some staining with the secondary Ab only which is not the case. So, are there any others explanations?
Thank you!!!
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