Fluorescence & Enzymatic Labeling for Tissue Components

Strictly speaking, fluorescence labeling isn't "staining" at all, but it's a common technique that can be used to identify specific subcellular components and even individual molecular species. It's not as intuitively understandable as staining is, so a bit of explanation is in order before discussing the example presented here.

This schematic diagram illustrates the principle of immunolabeling, which is used with either a fluorescent label, or one that deposits a dense colored material visible in ordinary light. There are two antibodies involved: a primary Ab against the molecule of interest, and a secondary one, which carries the "tag" against the first. Let's say, for example, you want to label for a certain glycoprotein in the basement membrane of the glomerular capillaries of a rabbit kidney (that's what the example is). You would get an Ab made in a mouse against the rabbit antigen (the glycoprotein). Then the secondary Ab would be against mouse antibodies. After you've reacted the primary Ab with the tissue, you react the secondary Ab with the primary, and the visualizable label is firmly attached at the location where the antigen is found.

Now, to return to the example above: at left you see a renal corpuscle with its glomerular capillaries in a standard H&E preparation, as an orientation. At right, you see a similar sample in which this indirect antibody labeling method has been carried out. The "tag" in this case is a fluorophore that glows when illuminated with ultraviolet light. The most common fluorophore is fluorescein isothiocyante (FITC), which emits a greenish glow upon UV irradiation.

Note that in the right hand image, some areas of the basement membrane are illuminated; some aren't. These are areas where the antigen of interest is present. Areas where there's a lot of it glow brighter than those where there isn't much; and in most of the glomerulus there's none, so you don't see anything. The net result is a ghostly outline of the capillary basement membranes.

Nor does the label have to be fluorescent. At left is an example of labeling using as a "tag" not a fluorescent molecule like FITC, but instead an enzyme. This enzyme is horseradish peroxidase, which is attached to the secondary antibody. After the initial reaction of primary antibody and tissue has taken place, the tagged secondary Ab is introduced as usual. At that point, the peroxidase is given a chance to work on an artificial substrate (in this case, diamino benzidine, or DAB) and it produces a colored precipitate at places where the label has "stuck."

This specimen is the endometrial lining of the uterus in a dromedary camel. The localization was done for the protein relaxin, a hormone associated with pregnancy and parturition. As you can see, the reaction is strongly positive in the lining epithelium (areas of dark brown) but completely negative in the underlying connective tissues. This distribution pattern confirms that relaxin is found only in certain places in this organ; and appropriate studies at different stages of pregnancy can clarify its presence or absence temporally as well.

Both these methods provide information about tissue structure and cellular activity that can't be obtained in in any other way, and variations on them exist for the electron microscope as well. They've become even more important and flexible in the past two or three decades as commercially-available antibodies to almost any antigen of interest have been made available. Immunolabeling with FITC and peroxidase is especially important for localizing gene products associated with various forms of cancer, and many of these routines have become standard diagnostic methodology.

I am grateful to Dr. Amal Arafat Mouktahr of the Suez Canal University for use of this image.

| H&E | PAS | Masson's CT Stain | Verhoeff-van Gieson | Verhoeff-Masson | Mallory's CT Stain | Golgi Stain|

Close this Window