Chasing Rainbows

How to make the most of multicolor immunofluorescence

Melissa Lee Phillips
Dec 1, 2007
<figcaption>Quantum dot immunofluorescence and nuclear cell staining of macaque dopaminergic neurons in the substantia nigra. The right image is a composite of the three markers. Credit: Courtesy of Pok Man Mendy Chan, Mount Sinai School of Medicine, New York</figcaption>
Quantum dot immunofluorescence and nuclear cell staining of macaque dopaminergic neurons in the substantia nigra. The right image is a composite of the three markers. Credit: Courtesy of Pok Man Mendy Chan, Mount Sinai School of Medicine, New York

Gone are the days when "multicolor immuno-fluorescence" meant proteins tagged with dyes of red, green, and blue. Today's fluorescent molecules cover the color spectrum, and researchers are using them to image proteins simultaneously.

Antibodies can be conjugated to two types of fluorescent molecules: organic fluorophores, or dyes, and quantum dots. Dyes, which link to antibodies that directly or indirectly recognize desired proteins, have been a staple in immunfluorescence studies for decades. That means many antibodies are commercially available and protocols are versatile. But dyes have drawbacks for multiplexing. An activated dye emits a wide spectrum of light...