We have several fixed coverslips of NK cells synapsing to cancer cell lines, which we were going to use in an experiment with demo equipment. The cells are labeled with Dil, but during the experiment, it became apparent that this dye was bleeding through into the green channel. I'm not sure whether the instrument just had poor quality filters or if this is just the dye itself, but in any event, we'd like to possibly use these slips for confocal imaging to look at the immune synapse.
Even if the Dil will stay in the red channel on a better imaging system, most of our conjugated antibodies are bound to Alexa 488, 568, or 594. We'd need to stain with 2 colors, and the dil will interfere with both 568 and 594 on our system. Does anyone know of a way to remove the dye? Or can we just blast the slides for a while/leave them out under the lights to bleach the old dye, and re-stain with whatever we want?
I'd like to avoid having to buy a blue fluorophore-conjugated Ab if possible, as we'd like to keep using DAPI for counterstaining. If anyone has any advice, it would be greatly appreciated!
P.S. I'd like to avoid making new coverslips at this point, as we've been having trouble culturing these (transduced) NK cells and our supply is dwindling -- otherwise the obvious solution would be to just re-make the coverslips!
Dear William,
If the problem is that DiI contaminates the Alexa 488 signal you could try to 1) increase the A488 labelling intensity, so that the DiI contamination can be tolerated, 2) subtract the DiI signal from the A488 channel by measuring the percentage of bleed-through in DiI-only control samples, or 3) lower the A488 excitation wavelength and narrow the A488 emission filter.
I do not think that you can use either Alexa 568 or 594 in conjunction with DiI, since the spectra overlap too much. If you need another colour, you may have to re-consider the blue part of the spectrum (or far-red).
Given that DiI is very lipophilic it will be almost impossible to remove it from your samples. In future experiments, you could reduce the DiI labelling (concentration/incubation time), so that the bleed-through will also be lower. I do not advise to try the bleaching approach: DiI will take a long time to bleach and you will most likely fry your epitopes so that subsequent immunolabelling will not work!
Hope this helps,
Chris
- Badges
- Science topic
- Similar topics
- Chemistry
- Biochemistry
- Biochemical Techniques
- Blotting Techniques
- Immunoblotting
- Dyes