[Histonet] Re: IF colocalization with 2 rabbit antibodies

Johnson, Teri TJJ <@t> stowers.org
Thu Dec 3 12:49:29 CST 2009

Hi Sam,

You asked if it was possible to do colocalization IF staining using two rabbit primary antibodies, one for p-S6RP and one for p-44/42.

You will have to be the one to determine if it's possible to do this on your samples. In theory, everything is possible. ;-)

First you'll need to optimize each of the antibodies singly in your mouse tissue, using proper negative controls substituting normal rabbit IgG and determining if you are indeed getting specific signal from each antibody. Once you have done that and you know what the antibody expression is for each one, you can move to designing the protocol for double staining.

There are a variety of ways to accomplish this you can find doing a literature search.

Probably the easiest method is to use a sequential IHC protocol where you treat with rabbit primary antibody #1, Fl-labeled secondary anti-rabbit #1, and then close any open binding sites using an incubation of unlabeled monovalent Fab fragments (http://www.jacksonimmuno.com/Catalog/monovalent.asp) or normal rabbit serum. Then you move on to rabbit primary antibody #2, and then Fl-labeled secondary anti-rabbit antibody #2.

Proper controls of this are critical to ensure what you are seeing is specific. Ideally, you will need 2 controls where you substitute normal rabbit IgG for one of the rabbit primaries but not the other (should end up looking EXACTLY like your single stain results), and another that substitutes normal rabbit IgG for both the primaries in the technique just as outlined above (should be totally negative).

If this isn't clear, don't hesitate to contact me directly. Another wonderful contact is Chris van der Loos, he does this all the time and makes it look easy.

Best wishes,

Teri Johnson, HT(ASCP)QIHC
Managing Director Histology Facility
Stowers Institute for Medical Research
Kansas City, MO

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