Hi everyone!
Dose any one knows how I could get rid of the cells fluorescent background?
I am working with two cells, HEK293 and SHSY5Y, and transfected them with exosome containing a protein-tagged with either EGFP (green) or mCherry (red). However, when I am observing the cell through confocal microscopy, I see the back ground in both cells either red or green and I am not be able to detect my exosomes-containing protein of interest.
Is there any suggestion/solution to remove the back ground to just detect my exosomes-containing protein of interest?
Best regards,
Farhang
Are you sure it’s background? It might look like background but it may actually be your vesicles in the cells, or the proteins are in the cytoplasm. Most cells don’t have that much fluorescent background that it would drown out signal in confocal microscopy.
what magnification are you using? Try a higher magnification, maybe you can’t see the vesicles properly if it’s too low. You could also try transfecting cells with untagged versions of these proteins (if they aren’t natively expressed) and staining for them to see if they are ending up in the cytoplasm, if you want to prove to yourself that it isn’t background and is just protein expression and unexpected localisation.
best of luck. It will work out. You’re doing a great job. Sometimes in science the result is not what you expect even remotely, but you learn something new and interesting.
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