I am doing IF on methanol-fixed cells (The antibody does not recognize the antigen if I use PFA to fix cells). I get high 'perinuclear' background even in control (only fluorescent secondary antibodies). If I use 100× lense, under confocal microscopy, the pattern of the background is clear and resembles mitochondria! The pattern is very clear and is even better than mito-tracker!!??? How could this happen? If I change my secondary antibodies to highly cross-adsorbed one, might it be useful to eliminate such background?
By the way, I am responsible for what I am saying and I have repeated this for three times. If this is real, it is very interesting about the 'target' of secondary antibodies inside the mitochondria, isn;t it?
I would suggest using secondary antibodies conjugated with another fluorochrome.
If you use rhodamine as a fluorescent conjugate to your antibodies, there may be some free rhodamine in the solution of secondary antibodies. Rhodamine may get inside mitochondria, and they give the signal you observe. The secondary antibody has nothing to do with it.
Hi, Elena
I didn't use rhodamine conjugated secondary. Actually I used both goat anti-mouse alexa594or 488, goat anti-rabbit alexa 594 or 488 and I can always get that signal pattern. I guess this phenomena is possibly cell line-specific...possibly.
Hi Yenddy,
I didn't permeablize cells with triton or something else. I only permeablize with 0.5% triton-x on PFA-fixed cells.
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