In molecular biology, the location of specific molecules in organelles and cells at the microscopic level has gained prominence in recent years through the development of a wide range of cytochemical techniques. These techniques are based upon the property that specific markers can be chemically coupled to a wide variety of molecules. Examples are fluorescent markers for light microscopy and electron-dense markers, such as ferritin or colloidal gold, for electron microscopy. The use of poly- and monoclonal antibodies has widened the possibility of localization to essentially every particle that can invoke an immunogenic response. The antibodies produced can be bound to markers used in microscopy and the distribution of the molecules of interest visualized directly by 1, 2-, or 3-step labeling procedures (1).
Immunocytochemistry is the application of antibodies in labeling protocols and must be performed in an aqueous environment to ensure that the antigen detectability is accurate and reproducible. Hence, cryotechniques are much preferred over the classical technique of chemical fixation, dehydration, and plastic embedment, which usually results in a loss of antigenicity. Along with cryofixation and cryosectioning, the use of colloidal gold is now commonplace. Colloidal gold has numerous advantages, among which are (i) it is electron dense and easily visualized in electron microscope images; (ii) various sizes of gold particles can be obtained, allowing double-labeling experiments; (iii) it can be easily complexed with a wide variety of ligands; and (iv) the maintenance of bioactivity of complexed molecules is excellent. Typically, the primary antibody binds to the antigen of interest and is visualized by labeling with a secondary antibody to which colloidal gold is conjugated in analogy to the use of fluorescent antibodies in immunofluorescence. With immunogold labeling, certain considerations should be addressed before starting (2). (i) Where is the antigen located? Additional preparative steps may be necessary to make the antigen accessible to the gold antibody. (ii) What is the nature of the antibody? A monoclonal antibody may not bind the antigenic site if the site is modified during electron microscope preparation; this problem is generally much less severe with cryotechniques. Polyclonal antiserum, which contains a number of different antibodies directed against more than one epitope on the antigen, is less susceptible to conformational changes or modification of the antigen. (iii) From which species were the antibodies derived? Care should be exercised to ensure that antibodies are raised to pure undenatured antigens in a species compatible with the labeling system.
A recent advance in photooxidation has allowed for precise correlative immunolocalization on the same specimen using fluorescent light and immunoelectron microscopies (3). With use of this technique, proteins can first be localized to individual cells or tissue types at the light microscope level with an antibody containing a suitable fluorescent label such as eosin. Subsequently, the protein can be further localized at the electron microscope level by illuminating the fluorophore in the presence of oxygen and diaminobenzidine. Fluorophores with a relatively poor quantum yield, such as eosin, produce reactive oxygen intermediates that oxidize the diaminobenzidine, producing a brown osmophilic polymer. Preparation of these specimens for electron microscopy by osmium fixation yields a highly localized osmium stain at the antigen. Fluorescence photooxidation can also be used with nucleic acid sequences by using biotinylated probes and an eosin-streptavidin conjugate (see Streptomycin).
