Question

IMMUNOLOGY

You are asked to provide your input in the following experiment performed by a colleague or yours during your summer internship. Jackie performed an immunohistochemistry experiment to determine the presence of IL-1b an important pro-inflammatory cytokine in human lung tissues. She used a primary mouse anti-human IL-1b antibody at a 1:1000 dilution, after washing she incubated tissues with the secondary antibody goat anti-rat biotin conjugated.

She is puzzled as her staining did not work at all. What recommendation would you suggest?

What other information would you gather from Jackie to assist her in her experimental design?

Answer 1

The staining did not work because the secondary antibody used is incorrect, The secondary antibody should be reactive to the primary antibody, i.e., mouse anti-human antibody. Hence, the secondary antibody would have to be goat anti-mouse biotin cojugated, instead of goat anti-rat biotin conjugated.

I would gather information about what Jackie is using as controls for her immunohistochemistry experiment. The use of proper control is very important to get comparable and correct results.