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I am a little bit ashamed, but I have a very basic question to put you: when we
are doing co-localization studies (IF) both primary antibodies should not be
raised in the same species. Why? What would be the result? I would like to read
a little bit more about this subject, where should I look for?

Thank you,

Fátima<<

Fatima - you can do this easily if you have abundant protein and directly labeled antibodies. However, if you are using unlabeled primary antibodies, you need a secondary antibody to complex with the first which adds either an enzyme, biotin, or a fluorescent label. The secondary antibody is usually targeted against the animal species the first antibody was made in. Therefore, if both your primary antibodies are rabbit, and you use a green fluorescent labeled anti-rabbit and a red fluorescent labeled anti-rabbit, both labels will bind with the rabbit species. Everywhere both antibodies bound with the target protein will label with both red and green.

Even though that will show colocalization, it does not give you the option of seeing each color channel separately. What if your antigens do not colocalize in every cell? It will look as though it does by doing your immunostaining this way. If primary antibody #1 is located in cell type A and cell type C, and primary antibody #2 is located in cell type B and cell type C, they should only co-localize in C. But by doing it with your method, you will also show colocalization in cell types A, B, and C.

Does this make sense?

If you use two primary antibodies raised in different species, say goat for Antibody #1 and rabbit for Antibody #2, you can use an anti-goat green fluorescent labeled secondary antibody, and an anti-rabbit red fluorescent labeled secondary antibody. The anti-goat should localize your Antibody #1, and the anti-rabbit should localize your Antibody #2. Therefore, given the above example, Antibody #1 will show positivity in cell type A and C, Antibody #2 will show positivity in cell type B and C, and they will only co-localize in cell type C.

For more information on multiple immunostaining methods, I urge you to purchase and read (cover to cover) Chris van der Loos' book, ISBN 038791594X.

Best wishes,

Teri Johnson
Stowers Institute for Medical Research
Kansas City, MO