All immunocytochemical techniques are based on a similar principle of incubating the target antigen with an appropriate antibody solution, but they may incorporate one or more from a range of dif ferent microscopically dense markers for visualizing the sites of binding. Light microscope immunocytochemistry was initiated by the classical work of Coons and co-workers in 1950, who developed the immunofluorescence technique for antigen localization (1,2). This was followed some time later in 1966 with the introduction by Nakane and Pierce of the immunoperoxidase procedure (3), after which there have been various attempts to increase the sensitivity of techniques for localizing tissue antigens, including peroxidase antiperoxidase (4), avidin biotin complex (5), and alkaline phosphatase anti-alkaline-phosphatase (6). These techniques are all still routinely used for immunocytochemistry, and the various advantages and disadvantages of each procedure that must be considered when choosing an appropriate procedure are addressed in Table 1. A recent review has discussed the major advances that have been made more recently in immunocytochemistry, which include increasing in label diversity, improving method sensitivity, and multiple labeling (10).
During the past two decades, it has been possible to use colloidal gold probes for immunolabeling at the light microscope level. Gold spheres, usually between 1 and 15 nm in diameter and in colloidal suspension, are coated with an immunological protein. As in the immunoenzyme techniques, the immunological protein could be one of a wide range of proteins. It could be a primary antibody, for use in a one-step system, or a secondary or tertiary antibody, for indirect labeling. It could be protein A, protein G, or, if an avidin-biotin system is used, streptavidin or an antibiotin monoclonal antibody could be coupled to the gold. Immunolabeling is conducted by incubating the antigen with the primary antibody-gold complex in the direct technique or primary antibody followed by the gold conjugate in the indirect technique. Strong immunogold labeling may sometimes be observed as its natural red/pink color under the light microscope, but frequently the labeling is too weak to detect unless a further enhancement stage is used. In 1983, Holgate et al. (11) described a novel technique that is applied in essentially the same manner as immunoenzyme staining, but involves the use of colloi-
Relative Merits of the Various Available Immunolabeling Procedures
Indirect immunoenzyme procedures.
New build-up immunoenzyme mogens. approach based on older method (7,8).
Immunoenzyme sandwich procedures such as peroxidase anti-peroxidase and alakaline phosphatase anti-alkaline phosphatase.
Colloidal gold labeling.
Enzyme polymer procedures.
Increased sensitivity second antibody procedures(9)
Rapid; simple procedure. Multiple labeling is possible using different filter combinations. Confocal microscopy may be used.
Reasonably rapid. Bright signal against a dark background is very sensitive. Many primary antibodies may be incorporated into the common procedure. Multiple labeling is possible using different filter combinations. Confocal microscopy may be used.
Rapid. Permanent preparations. Simple nuclear counterstains may be used for studying tissue architecture in relation to immunolabeling.
Reasonably rapid. Permanent preparations. Simple nuclear counterstains may be used for studying tissue architecture in relation to immunolabeling.
Highly sensitive. Useful for retrieving technical failures in immunocytochemistry.
Relatively rapid. Sharp, black, high-resolution reaction product. Sensitive. Permanent preparations. Nontoxic reagents. Can be studied using various forms of microscopy including dark ground, epipolarization, confocal laser scanning and electron microscopy.
Extravagant when more than just a few target antigens are to be studied.
Fading of fluorochromes. General tissue architecture is not visible. Preparations not permanent. Fluorescence microscope required. However, newer, brighter fluorochromes are more resistant to fading, especially when used with anti-fade mounting media.
Use of toxic chromogens is involved. Need to block endogenous enzyme activity. Extravagant when more than just a few target antigens are studied.
Use of toxic chromogens is involved.
Need to block endogenous enzyme activity.
Extremely laborious. Uses toxic chromogens. Need to block endogenous enzyme activity.
Laborious. Uses toxic chromogens.
Need to block endogenous enzyme activity.
Laborious. Toxic chromogens. Need to block endogenous biotin activity. Prone to fading. Capricious in inexperienced hands. Temperature-dependant.
Kits are relatively expensive.
dal gold-labeled antisera that are strongly visualized for light microscopy by silver enhancement (Fig. 1). This method is called immunogold-silver staining (IGSS) and makes use of the Danscher physical developing solution (12) to create a layer of black, metallic silver over the gold-labeled binding sites. Table 2 describes the historical progression of the IGSS method. Danscher's reagent is a mixture of silver lactate (the silver ion source), hydroquinone (the silver reducer), and gum acacia (a colloid that prevents rapid, auto-reduction of the silver lactate in solution). A range of easy-to-use, commercial alternatives to this mixture are also now available because IGSS is now an established procedure with a role both at the light microscopy and the electron microscopy level (see Note 1).
Historical Development of IGSS
First mention of gold-protein complexing and interaction with light.
Faulk and Taylor (14)
Electron microscopy of gold probes established.
Silver probe for gold detection in tissue sections.
Adaptation of the Danscher procedure for IGSS.
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