Double immunofluorescence staining (cyto) - anti-mouse and anti-rat, too similar? (Dec/02/2005 )

Hei all!

I am very newbie on the area of immunostainings, and now I have a huge problem. Mayby some of you, more experienced scientists, could advice me a little?

What I am trying, is to stain mouse cell culture with two different primary antibodies ( one produced in mouse and one in rat), so then I used secondary fluorescent antibodies against rat and against mouse.
interestingly, it seems that primary antibody is cross-reacting with secondary antibody in rat. Do you have any experience on this matter? Are these two species too close to each others or this is a single phenomen related to my secondary anti rat antibody, which was not purified against croos reaction to mouse serum?

This is the information that is given about purification of rat secondary antibody:

http://www.chemicon.com/Product/ProductDat...ductItem=AP189R
Antibody was isolated from whole goat antiserum by antigen affinity chromatography. Monospecific by IEP against whole rat serum. Reacts with heavy chain of Rat IgG, as well as the light chains from most rat immunoglobulin classes.

To ensure minimal cross-reactivity to non-target immunoglobulin, the antibody was solid-phase absorbed against serum proteins from bovine, chicken, goat, guinea pig, hamster (Syrian), horse, human, rabbit, and sheep.


Millions of thanks to all of you, who had time to read this issue

Hi Elmis

I'm not an immunologist but I had the same trouble once and what we concluded was that anti-rat conjugates prepared against the whole IgG molecule are simmilar enough to this of mouse to cause cross reaction. So maybe what you could do is to get a conjugate specific to the Fc of the IgG which should be more specific for rat IgG. Also you could try labelling with your mouse antibody, then with the proper conjugate, then with the rat antibody and finally its conjugate, with several washes between each other, so that label of the antibodies with theri correpondant conjugates will be more accurate.

Hope it can help you at least a little bit

Good luck,

Pipo

Yes mouse and rat Fc regions are very similar.

Got to JacksonImmuno.com and buy a goat or donkey anti-rat IgG which has been cross adsorbed against mouse. For example this one is a personal favorite of mine Catalog # 712-065-153. Their antibodies are excellent and it works like a charm even to stain mouse tissue sections without loss of appreciable signal.

(incidentally they come in all sorts of formulations also with Fab fragments and Fc specific in case you need it)

Thank You all a lot