Seeing signal from Alexa Fluor 594 under 488 excitation ?

14 April 2021 5 1K Report

I am currently working on protein co-localization with confocal microscopy with 2 proteins, let's call them protein A and protein B. Protein A is located mostly in cytoplasm with little amount in the nucleus while protein B is found in the nucleus only.

So my experiment has 2 parts - first, I need to treat the cell with a certain drug so that protein A is entering the nucleus. For this part I stain the cell with anti-protein A antibody and also Alexa Fluor 594, then counterstain with DAPI. I am using Leica confocal mic which has the typical fluorescence microscope functions (with DAPI, GFP and RHOD filters). Before proceeding to the confocal functions, I checked the cell with the typical fluorescence microscope function to make sure there's signal appearing from the samples. When I use the GFP488 filters, I noticed some signal coming from the samples even though I did not stain anything related to 488 (only alexa fluor 594 and DAPI). I didn't bother as I think alexa fluor 594 and DAPI are so different and I just ignore it. The result is beautiful at last and I am seeing protein A inside the nucleus after treatment.

With protein A inside the nucleus, I started investigating the co-localization between Protein A and Protein B. When I single-stained the sample with anti-protein B antibody and also alexa 488, I saw some weak signal from the cytoplasm (but protein B is a nuclear protein), but I could still manage to take acceptable photos showing protein B from the nucleus and not a lot from the cytoplasm. The problem comes when I stain all DAPI, Protein A and Protein B. I am using anti-protein A and anti-protein B antibody with alexa fluor 594 and 488 respectively. This time when I view the channel at 488 I see a lot more signal from the cytoplasm (note protein B is a nuclear protein).

Remembering that I have signal seen from 594 when using 488 filters I was thinking if it's possible that I am seeing part of the signal from protein A. I heard that when 2 fluorophores have big intensity difference the signal from one fluorophore could possibly spilt to another channel.

The samples I have here is pretty valuable so I am guessing if this is the case I might just strip off the antibodies and re-stain.

Any suggestions?

Debadrita Pal

To the first part of your question, I would say that I am not sure f you are aware of a phenomenon called "bleed through", it happens especially with Dapi. I usually do 1:10000 Dapi. Make sure you are not using a lot of Dapi and are diluting it appropriately, otherwise high possibilities that it can bleed through into the green channel.

To the next part of your question, it sounds like you might be getting a bleed through in here as well, I would suggest trying the experiment without the Dapi once.

I am not entirely sure what samples you have so not sure if you can strip off dapi.

Bertha Mak

Hi Debadrita Pal thanks for your help. I am using fixed cell and the cell line is Hela.

So DAPI, anti-protein A with alexa 594 and anti-protein B with alexa 488.

Debadrita Pal

Hi,

I understand the samples are valuable. But I am afraid that I am not aware of stripping off Dapi from fixed Hela cells. And reading your situation I think you would have to re do the fixing again. I would this time try and first stain for protein A as well as B but not Dapi. Or lower Dapi concentration.

(I usually do 1:10000 dilutin for Dapi, and bleed through is a very common phenomenon with Dapi).

Radek Machan

Hi, since you are using a confocal, you should be able to combine freely different lasers and different filter sets, so you can play with this to find more. In this way you can test whether the green fluorescence is excited by the laser for DAPI (405 nm) or by the laser for green fluorophores (488 nm or similar). DAPI has fluorescence stretching far into green, so you would definitely see it with green emission filter if you excite with 405 nm, on the other hand DAPI is not excited by 488 nm laser. I'm not sure how you are acquiring the images, but you should definitely try to avoid using all lasers simultaneously. Take an image with each laser individually to minimize bleed through between the channels. The confocal should have functions to do this automatically. You may switch the lasers for each line of the confocal scan, so the images in all channels are acquired nearly simultaneously (though for fixed samples this doesn't really matter as you don't expect things to move).

Bertha Mak

Thanks for all of your help !! Some bit of a updates:

- hmmm I guess it is the primary antibody which is not a good enough and it's probing elsewhere in the cytoplasm... It got a bit better when I increase the % blocking and now it looks acceptable ( still a bit signal from the cytoplasm tho)

- I tried to adjust the detector (to narrow the emission wavelength it detects) and it also gets better

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