Dear Marina,

I strongly recommend you not to use such combination, since both your secondary anti-rabbit antibodies (although with a different color) will probably bind to both your Iba-1 and ninjurin-1 abs. You will probably get contaminated signals. You should use different primary ab (different species) and respective secondary ab. That’s what I would do. Good luck!

Hi Marina,

One possibility is to convert the primaries into a directly labelled probes by pre-conjugating them to the secondaries. There are kits available for this but I haven't used any so I cannot recommend anything.

As mentioned by Jose, using two primary antibodies raised in the same host is not compatible with the standard indirect immunofluorescence approach to immunolabeling. You will get beautiful false-positive immunolabeling.  However, there are a variety of ways that two primary antibodies raised in the same host can be used together.

One approach, using primary antibodies that are directly tagged with different fluorescent labels was mentioned by Matthew above.

Other approaches that can work involve sequential labeing of the tissue with the first primary (Iba-1) and fluorescently tagging it, then labeling the tissue with the second primary antibody (ninjurin-1) and fluorescently tagging it using a tag with a different wavelength from the first tag. One approach to this would be to use a fluorescently tagged Fab fragment secondary antibody to visualize your Iba-1 labeling. The Fab fragment antibody has only a single binding site and, once bound, could not interact with your Ninjurin-1 antibody when it is applied later. Critically, you must use this Fab Fragment antibody in excess, so that it will bind up all the available Iba-1 antibody. If the Fab fragment antibody does not occupy all of the Iba-1 antibody, you will get false-positive double labeling when the next fluorescent antibody is applied. After completing labeling for Iba-1 and the Fab fragment secondary antibody, you would then apply your Ninjurin-1 antibody to the tissue and visualize it using a secondary antibody with a fluorescent tag in a different fluorescent channel from the Fab antibody (for instance, you might select a goat anti-rabbit Fab-Cy2 antibody for visualizing Iba-1, and a goat anti-rabbit- Cy5 antibody for visualizing Ninjurin-1).  These approaches are ticky and require very careful titration of the antibodies, as well as other controls to be certain that any double-labeling you observe is not due to false-positives. Its a pain, but it can work.

   There are several other variations on this theme. The Jackson ImmunoResearch Website offers descriptions of several of these approaches, and also sells Fab fragment antibodies that can be used in these methods.

   Another approach would be to label your tissue for Iba-1 and image it, strip that labeling, then label your tissue for ninjurin-1, go back to the microscope and image Ninjurin-1 labeling in the same region of the specimen where you imaged Iba-1.

   After all that is said and done, it would be a whole lot easier (and probably cheaper in the end) to buy a different  Iba-1 primary antibody raised in goat or mouse. There are several available on the market. Hope this helps. Good luck.

Never use two antibodies from same host for multi staining You will get false signals only. buy new antibody.