Autofluorescence problem

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aes
aes's picture
Autofluorescence problem

Hello,
I actually observe B-cell by confocal microscopy. I label a membrane protein with Alexa Fluor 488 and when I observe them in green channel (Excitation 488 nm, Em 520 nm) I see a strong autofluorescence as granules in the cytoplasm! Does anyone ever met this problem?
Thanks you in advance.
Bye.
Manu.

Fraser Moss
Fraser Moss's picture
You can eliminate/greatly

You can eliminate/greatly reduce these autofluorescent blobs by giving a brief spin of the label (up to 1 min) in a microcentrifuge or a picofuge (top speed e.g 14000xg) before you dilute it into the sera for staining. The spin brings all the non conjugated debris to the base of the tube (which you can see as a blob of dye) and you take your sample from the liquid phase at the top for dilution.

aes
aes's picture
I will try it! Thanks a lot.

I will try it! Thanks a lot.

blackmouse
blackmouse's picture
I am staining for membrane

I am staining for membrane proteins on immune cells with FITC fluorescent a.b.
doing my negative controls  I also see a lot of little green particles in the cytoplasm (Excitation 488 nm, Em 520 nm).
I was wondering if you tried to centrifuge the a.b. and if it worked??
 

heehawmcduff
heehawmcduff's picture
I'm just wondering if the

I'm just wondering if the little green blobs that you see are actually in the cytoplasm or are membrane bound on the apical surface of the cell?