Hi Hatoon,

1) You can check for autofluorescence first (same procedure without antibodies and (strep-)avidin. Check both green and red channel. This is commonly more prominent in the fitc channel than in the others, however erythocytes will also fluoresce in red channel as well but are easily to find do the morphology. If this is the problem, shifting to another fluorophore is the easiest way.

2) Does your secondary bind without primary? It might help using secondary antibodies that are pre-absorbed to omit cross-reactivity with other species.

3) Pre-blocking with higher concentrations of serum might also help for non-specific binding of the antibodies.

4) If you are using avidin-biotin system, you might have to block with free Avidin and Biotin.

Be sure that your ephB4 antibody doesn't react with other partners. We checked several ephB2 antibodies, and they were not specific:

Article Investigation of Cross-Reactivity of Anti-Ephrin-B2 Antibody...

BW

Olav