The prototypic Th2 cytokine IL-4 is essential for the development of Th2 responses, as it promotes the differentiation of T helper cells into type 2 effector cells and inhibits type 1 responses. The expression of this important cytokine is very tightly regulated, and IL-4 is also at very low quantities compared to other cytokines (it is expressed 1000-fold lower than its Th1 counterpart, IFN-gamma), making its detection extremely difficult1. In an attempt to examine IL-4 expression in vivo, recent studies have employed IL-4 reporter knockin mice, where an IL-4 promoter drives the expression of GFP to identify IL-4 expressing cells, and have described IL-4 expression by basophils and eosinophils, as well circulating T cells2-5. Although quite useful, potential caveats with this system are that expression of the GFP may not be similar to IL-4 protein, or even reflect the cytokine's mRNA levels, as the GFP is processed differently by the cell and has a much longer half-life. An additional shortcoming of this approach is that it is not readily useful for in situ imaging.
To directly examine IL-4 protein expression patterns in situ, we developed a new methodology, where two antibodies recognizing distinct epitopes of the IL-4 proteins were used in tandem. These antibodies were conjugated to the same fluorochrome, resulting in amplification of the fluorescent IL-4 signal enough to be detected by conventional fluorescent microscopy. Additional antibodies conjugated to different fluorochromes can be added to the staining cocktail, to identify distinct leukocyte populations, etc, and these images merged with the IL-4 images.