chapter  Chapter 5
36 Pages

Immunofluorescence

WithAnthony F. Henwood

Polyclonal antibodies were the first immunoreagents used resulting in unprecedented strides in biology. Animals commonly used for antibody generation are rabbits, goats and to a lesser extent hamsters, rats, and mice. A polyclonal antiserum contains a mixture of different antibody isotypes as well as nonsense immunoglobulins that are normally present in the rabbit serum. Natural antibodies binding to the tissue section may occur in the antiserum as a result of unrecognized, prior antigenic stimulation. Endogenous Fc receptors are structures on the surface of certain cells that bind the Fc region of antibodies. Evidence that the antibody binds specifically to the expected target molecule is often provided by a Western blot, which should show that the antibody stains a single band of appropriate molecular mass for that target. Plant and animal tissue contain naturally fluorescent compounds. The fluorescence phenomenon itself is based on the reaction of formaldehyde vapor with intracellular biogenic amines such as dopamine, epinephrine, and norepinephrine.