Immunocytochemistry (ICC) — Post #3

Immunocytochemistry is the backbone of my research and for the last two weeks I have been learning the procedures and methodology behind this important piece of my work here this summer. One of the possible heritable difference between reproductively responsive and non-responsive mice is the difference in the number and activity of GnRH neurons involved in initial stages of the HPG (Hypothalamic-pituitary-gonadal) axis. The way that we see the difference between neurons in the reproductively responsive or non-responsive mice is through immunocytochemistry or ICC. ICC uses engineered antibodies to bind to proteins of interest.

The primary antibody used to detect the mature GnRH protein is called SMI-41. During an overnight incubation, SMI-41 reacts with the “five amino acids adjacent to the C-terminus of the GnRH peptide and the amidation site,” (Avigdor, 2004).

After the overnight incubation in primary antibody, the brain sections were given a secondary antibody. This secondary is called biotinylated horse anti-mouse. This “anti-mouse” secondary antibody binds to the mouse primary antibody. A special characteristic of the second antibody is the biotin (hence “biotinylated”) located on the end of the protein.

The biotin on the secondary antibody binds to the avidin in what is called ABC or avidin biotin-peroxidase complex. This complex has an enzyme called peroxidase that undergoes a color change when reacted to peroxide and a substrate called DAB (3,3′-diaminobenzidine tetrahydrochloride). The color reaction turns wherever the peroxidase complex is bound a brownish color.

Once the color reaction occurs, the brain sections are mounted individually on a slide. The slides are inspected under a microscope. Under inspection in ~100x magnification, one may see the mature GnRH contained inside GnRH secreting neurons. The neurons are counted and a reliable measure of reproductivity can be assessed.

Mounted brain slices after ICC staining

Mounted brain slices after ICC staining