I am performing IHC in mouse brain slice. I am using two antibodies for the purpose of co-localization. My first target remains constant in all experiments and is a guinea pig primary. The secondary I am using to detect this primary is Donkey anti-guinea pig conjugated to Alexa-488. My second target is generated in mice, and the secondary I am using to detect this target is Goat anti mouse conjugated to Alexa-594. When I perform this experiment everything looks great the signals are separate, each channel looks different. Now the weird thing is when (keeping my first target constant_the guinea pig Ab) if I switch my second primary to another target (but is also mouse) and use the same Goat-anti mouse secondary-594; now all of a sudden it looks like both channels are completely identical and the signal I see in the green looks the same in the red channel. Why would changing primary antibodies do this??? I am missing something fundamental here? Thanks for your help?
Hi everybody
In my opinion, with Alexa488 (abs 496 nm; em. 519 nm) it would be better to use the Alexa594 (abs. 590 nm; em. 617 nm) than Cy3 (abs. 550 nm; em. 570).
I also think that, since when using the other primary antibody there is not cross-talk, the dye should not be the problem
Regards
First of all, I would perform a negative control without primary antibodies. I had problems in the past with an anti-mouse secondary antibody recognizing some structures of mouse tissue.
If the secondary is not the problem, then either the primary is binding to the same protein (I don't know what proteins you are working with, but then it may be a bad antibody) or it is binding to another protein that colocalize. In this case you can:
- Try with a shorter incubation period for that antibody, since sometimes too much exposure may give you inespecific binding.
- You can do a western blot to know if they are binding to the same protein (althought sometimes the binding in WB and in IF are different).
- Try with a different antibody for the same target, to know if it is just a bad antibody
Hi everybody
In my opinion, with Alexa488 (abs 496 nm; em. 519 nm) it would be better to use the Alexa594 (abs. 590 nm; em. 617 nm) than Cy3 (abs. 550 nm; em. 570).
I also think that, since when using the other primary antibody there is not cross-talk, the dye should not be the problem
Regards
What is the animal you are trying to stain? Mouse on mouse is a problem. I also would really consider whether or not double immunofluorescence is necessary. If one of the two molecules is in a different cellular compartment, I'd use immunoperoxidase for one (smaller compartment) and fluorescence (amplified) for the other. that way there is no bleed through problem, and the peroxidase substrates block any reagents from joining the complex.
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