I already tried blocking, which was fine for lines, but the B cells present so many antibodies that I cannot cover. The secondary antibody control still shows massive cytoplasmic/membrane staining. Any suggestions?
I think that is physically impossible, for the reason you already mentioned. The Immuglobulin attached to the B-cell surface is indistinguishable from your primary mouse antibody, if you use a general anti-mouse secondary. The most reliable workaround is to use a primary AB from a different species or directly conjugated antibodies.
If that's not an option, you could try a secondary only against the specific isotype of your primary AB (e.g. IgG2a). This should not stain all your surface IgD or IgM, at least not as strongly.
I think that is physically impossible, for the reason you already mentioned. The Immuglobulin attached to the B-cell surface is indistinguishable from your primary mouse antibody, if you use a general anti-mouse secondary. The most reliable workaround is to use a primary AB from a different species or directly conjugated antibodies.
If that's not an option, you could try a secondary only against the specific isotype of your primary AB (e.g. IgG2a). This should not stain all your surface IgD or IgM, at least not as strongly.
There are kits for this type of problem (staining mouse tissue with a mouse primary). They are sold as mouse-on-mouse kits (MOM), but you'll have to try if they work in your particular case. Theoretically it's possible to block all the endogenous Ig's, but at the bench theory often seems to be on holiday...
I've heard of good results with the kit Dako sells.
I fully agree with Jonathan, these mouse-on-mouse kits are very helpful and work well in immunohistochemistry. We had best results on paraffin-embedded material with the kit from Nichirei Bisociences, but as Jonathan mentioned, you have to try...
Label your primary using Mix n' Stain kit (Biotium), it's very cheap, 30' labeling, no wash of the unbound dye is needed and works beautifully...this way you don't need secondary ;-)
Here is the link: http://www.biotium.com/product/product_info/newproduct/Mix-n-Stain_Kits.asp
Good luck!
Agree with the above MOM kits can be useful but I would suggest conjugating your antibody to biotin or fluorescent dye directly using commercially available kits thus avoiding using anti-mouse secondaries all-together.
You may biotinylate your primary antibody and use fluorescence labeled streptavidin as secondery reagent or lable the primary ab directly with a suited fluorophore.
You can block those sites by incubation with excess (like 1:100-250) Fab fragment goat, rabbit, or donkey antimouse IgG (H+L) . [You can get these from Jackson ImmunoResearch.] Then wash and do your ICC. Wev'e done it, it works.
Woops! I forgot to mention obviously the fab anti Mouse IgG(H=L0 is unlabeled.
Is there a pre conjugated anti AID antibody available ? The point is that I want to use the PLA assay and only pretest the ab 's.I have to use two different ab's (e.g. mouse and rabbit) for that assay, that's why I need to block the surface ab's to avoid cross reactivity .
Thanks for all the good ideas !
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