Fixation on Histology

Fluorophoric vs Chromogenic Labeling


Fluorophoric vs Chromogenic LabelingIn immunohistochemistry, labeling refers to adding a “tag” to an antibody to aid in detection. There are two main types of IHC labeling, fluorophoric and chromogenic.

Fluorophoric uses compounds that produce a fluorescent signal when excited by UV light. This type requires darkfield microscopy for viewing. A little background on darkfield microscopy… Darkfield microscopy involves essentially blocking out the light that would normally be surrounding the specimen to create a dark background that allows for added contrast between the specimen and background. What is actually happening, is light is reflecting off only the particles on the slide thanks to a disc placed under the condenser lens. It is typically used for specimen that are transparent, or the same color as their surroundings. In IHC, fluorophoric labeling is typically used on frozen sections, not paraffin embedded formalin fixed tissue.

In fluorophoric labeling, the compound that produces the signal is called a fluorophore. There are several different fluorophores that are commonly used, including Fluorescein, which is one of the oldest and produces a green color, and Rhodamine (magenta). The downside to these fluorophores is that they fade pretty quickly when exposed to light. This is a big problem since the intensity of the stain is a big part of what you’re looking for in IHC. There have been newer fluorophores developed in the last ten years that are more resistant to fading, including Alexa Fluor™ 488 / Dylight™ 488 and Alexa Fluor™ 594 / Dylight™ 549, to combat this issue. The fluorophore is either coupled with the primary antibody (the direct method) or with a secondary antibody (the indirect method). Read our post on indirect vs direct IHC.

Chromogenic, on the other hand, relies on an enzyme/substrate reaction to produce a pigmented deposit, and can be observed using brightfield microscopy, which is your typical microscope viewing. In chromogenic staining, the compound is called a chromogen. Some chromogens include Di-Amino-Benzidine (DAB), Amino-Ethyl-Carbazole (AEC), Bajoran Purple™, Vina Green™, Fast Red (FR), among others. Polymerization of the compound occurs during the reaction between the enzyme, or the label being used, most often Alkaline Phosphatase or Horseradish Peroxidase and the substrate. This polymerization, and resulting deposit, take place at the antigen site. These products are commercially available so that you are guaranteed to have the right amount of enzyme, substrate, and chromogen so that you get quality staining.

Learn more about IHC labeling in NSH’s QIHC Prep Course, available on our online learning center,