Question

You have conducted an experiment to try to demonstrate that growth factor receptor X protein (GFRX) and ligand Y (LY) are expressed in the same cells using immunohistochemistry (IHC). You are using the following antibodies:

Primary antibodies:

Goat anti-GFRX

Goat anti-LY

Secondary antibodies:

Horse anti-goat-FITC (FITC is a green fluorochrome)

Donkey anti-goat TRITC (TRITC is a red fluorochrome)

As you view your experiment under the appropriate fluorescent filter sets, you show that both labels (red and green) are expressed in a subset of the total number of cells, but they are always co-expressed. To be sure, you’ve collected your images under a confocal microscope. Thrilled with your results, you start writing a paper to publish in the journal Nature. Should you? Why or why not?

Answer 1

The results should not be published.

Because the co-occurrence of red and green labels are due to experimental error and not due to actual co-expression of receptor-ligand in the same cells.

In the above experiment, both the primary antibodies used are raised in the same species, goat, which will share the secondary antibody binding sites where both anti-goat-FITC and anti-goat-TRITC will bind, resulting in the co-occurrence of both labels.

In IHC, while testing for co-expression, primary antibodies against each target should be from a different species. For example, the above experiment will work if the primary antibodies are

1. Goat-anti-GFRX primary antibody
2. Rabbit-anti-LY primary antibody

And hence the secondary antibodies should be

1. Horse anti-goat-FITC
2. Donkey anti-rabbit-TRITC