co staining problem: same primary species Options

[dendriet]

Hi all,

I'm kinda new to this forum, hope you might help me further. I want to perform co-staing with IHC.
One primary is mouse anti-CD54, the other is mouse anti-dextran FITC (directly conjugated, no seconday AB needed). My question is: how can I perform co-staining while the species of the 2 different antibodies are the same? Because, since my secondary for the mouse anti-CD 54 would be a goat-anti mouse Texas Red, this would react with the mouse-anti dextran FITC as well. How can I prevent this?

thanks in advance!

[mdfenko]

stain first with the anti-cd54 and the anti-mouse secondary then stain with the anti-dextran.

[dendriet]

stain first with the anti-cd54 and the anti-mouse secondary then stain with the anti-dextran.

Thanks! But don't you think the mouse anti-dextran will then bind to some of the free secondary goat-anti mouse parts which didn't bind to the mouse anti-CD 54? Shouldn't I saturate these 'free' seconday anti-mouse spots with some kind of serum?? Thanks a lot again..

[mdfenko]

Thanks! But don't you think the mouse anti-dextran will then bind to some of the free secondary goat-anti mouse parts which didn't bind to the mouse anti-CD 54? Shouldn't I saturate these 'free' seconday anti-mouse spots with some kind of serum?? Thanks a lot again..

there should be no free secondary anti-mouse. all of the unbound antibodies should be washed away after incubation with the secondary.

[dendriet]

Thanks! But don't you think the mouse anti-dextran will then bind to some of the free secondary goat-anti mouse parts which didn't bind to the mouse anti-CD 54? Shouldn't I saturate these 'free' seconday anti-mouse spots with some kind of serum?? Thanks a lot again..

there should be no free secondary anti-mouse. all of the unbound antibodies should be washed away after incubation with the secondary.

Ok, thank you. By 'free' I was actually referring to the free Fab part (variable part) of the secondary antibodies of which just one Fab part has bound to the Fc part of the primary. Or is it impossible for the secondary to have one Fab part 'empty'?? Thanks..

This post has been edited by dendriet: Nov 4 2009, 06:48 AM

[miBunny]

Why not just label your antibodies before staining? Invitrogen makes a kit for small scale antibody labeling. Its called either zenon or xenon labeling. Its pretty easy and I think it only took around 10 minutes. It was bloody expensive but it did work really well

[wirly]

Thanks! But don't you think the mouse anti-dextran will then bind to some of the free secondary goat-anti mouse parts which didn't bind to the mouse anti-CD 54? Shouldn't I saturate these 'free' seconday anti-mouse spots with some kind of serum?? Thanks a lot again..

there should be no free secondary anti-mouse. all of the unbound antibodies should be washed away after incubation with the secondary.

Ok, thank you. By 'free' I was actually referring to the free Fab part (variable part) of the secondary antibodies of which just one Fab part has bound to the Fc part of the primary. Or is it impossible for the secondary to have one Fab part 'empty'?? Thanks..

You can pre-assemble the two primary-secondary antibody pairs prior to adding to the cells. Incubate the primary and secondary together using an slight excess of secondary, then "quench" the secondary with a large molar excess of mouse IgG isotype control (something that will not bind to your cells) incubate far a bit. This mops up all of the free secondary fab binding sites. You can then stain the cells with the primary/secondary mixtures, probably even at the same time, though I might try them sequentially. Don't do really long incubations as this may lead to swapping of the secondaries. I second the xenon kit, they rock, they are a very refined version of what I just described.

[dendriet]

Hi all,

Thanks for the valuable input! Indeed I will try the labelling of my antibodies beforehand, I'm ordering the Zenon kit from Invitrogen already...eager to know what the result will be..
Thanks again for the willingness to help.