Immunocytochemistry antibody labeling methods

For direct immunocytochemistry, the label is bound to the 1° antibody. For example, to locate an antigen in cells, the 1° antibody would be a rabbit anti-antigen labeled with fluorophore (Fig. 7.1). Such a procedure with directly conjugated antibodies requires only the 1° antibody and no additional antibodies. Thus, the direct immunocytochemical procedure is the simplest method, and historically, was the first method for immunocytochemistry. 

Fig. 9. Calcium binding proteins in the MOB. A. Distribution of cells labeled with an antibody to Calbindin (CALB) using immunocytochemistry. B. Parvalbumin (PARV) immunocytochemically labeled cells are found mainly in the EPL in the bulb. C. Golgi stained section showing impregnated external tufted cells. D. An example of how cells may be identified by other methods. In this case an external tufted cell is shown after intracellular injection of the marker biocytin. Bar in D, /tm.

This new definition of immunocytochemistry derives from advances in antibodylabeling methods in recent years. These advances resulted from specific needs in animal research. Initially, formalin-fixed paraffin sections were used for immuno-histochemistry however, results were inconsistent. In most cases, the antibody did not label anything or it labeled too many cells and was dubbed over fixed. This problem led to the development of the epitope retrieval or antigen retrieval methods, where sections of tissue are treated with heat in buffers before antibody incubations. Unfortunately, epitope retrieval methods can be unique from antibody to antibody and also, for the same antibody, from tissue to tissue. Epitope retrieval is complicated and best avoided. For animal research, a simple method was then developed where tissue was fixed in paraformaldehyde and not formalin or alcohol and subsequently frozen sections were cut on a cryostat. This eliminated the steps of dehydration, embedding in paraffin, rehydration after sectioning, and epitope retrieval before antibody incubation. This was a major breakthrough. 

Immunocytochemistry harnesses antibodies that are specific reagents and which allow unique detection of proteins and molecules. Using antibodies requires specific methods, labels, and controls. Performing immunocytochemistry experiments requires some basic knowledge of biology. 

Morphological approaches in biomedical research can include a wide range of microscopes, but today typically employ immunocytochemistry that can give us information about individual liver cells containing the specific enzyme. Immunocytochemistry uses antibodies to bind proteins and labels to show protein s location. If, for example, the enzyme is a marker for inflammation, then the location of cells with this enzyme tells us which cell types have the inflammatory response. Thus, immunocytochemistry methods are broadly defined as individual studies of single cells or cell groups. The resulting data tell us about location of the enzyme. 

Another example of a population study that uses antibodies is flow cytometry. Isolated cells must be dissociated from tissues or cultures and labeled with fluorescent antibodies specific for a subpopulation of the cells. In flow cytometry, cells pass rapidly past a detector that measures the amount of fluorescence for each cell. The size of cells and the amount of fluorescence can be plotted and analyzed. Even though this method makes use of antibodies, it is a population study because it determines the number of isolated cells bound to an antibody. Flow cytometry identifies different populations of isolated cells, but it cannot show the location of these labeled cell in tissues, which can be done only with immunocytochemistry. 

Fig. 7.1 Direct immunocytochemistry. In the direct immunocytochemistry method, the 1° antibody binds its antigen and the label is directly attached to it...

A major advantage to the indirect method is that each labeled 2° will attach to all 1° antibodies from one species (e.g., goat anti-rabbit IgG labeled with 488 flu-orophore works for all rabbit 1° antibodies). This is the method of choice today because so many different antibodies are used in biomedical research. Indirect immunocytochemistry detects proteins in cells with the high detection sensitivity, good flexibility of reagents, and the fewest steps. 

One advantage of indirect avidin-biotin method is an amplification of the detection system. For example, after biotin conjugated 2° antibody, avidin is incubated, binding to the available biotins. After the remaining free avidin is rinsed off, a biotin conjugated to 488 fluorophore is added and it binds to the remaining sites on the avidin. With this method, amplification occurs because any avidin can be attached to multiple biotins conjugated with either fluorescent or enzyme. This method requires two additional incubation steps of the indirect immunocytochemistry with fluorescent-labeled 2°. 

To help evaluate possible labels and methods, use Table 9.1. Note that the number ratings in Table 9.1 are based on the author s experience and should only be used to compare methods within this table. Detection resolution is the ability to localize the label to the exact site of the primary antibody. The direct immunocytochemistry method does this and is rated as a detection resolution of 10 (Table 9.1, Fig. 9.1a, arrow). With detection methods that have lower detection resolution the number decreases (Table 9.1, Fig. 9.1a, numbers). 

Designing an experiment with a single 1° antibody involves many of the topics presented in the previous chapters, including reagents, types of label for antibodies, and the methods for applying antibodies. This chapter presents a step-by-step approach to designing an immunocytochemistry experiment and depends on information in the Immunocytochemistry Experimental Design Chart completed in Chapter 9. 

To use this Zenon method, the individual 1° antibodies are labeled individually with fluorescent-labeled Fab before the immunocytochemistry incubation with cells. To do this, incubate with the 1° antibody, mouse anti-Ag A, with anti-mouse Fab 488 fluorophore reagent (Fig. 12.4a) and the labeled fluorophore, which binds to the P antibody (Fig. 12.4b). Excess labeled 488 fluorophore Fab reagent must be present in the incubation solution (Fig. 12.4b). The blocking reagent, normal mouse IgG contained in nonimmune mouse semm, binds the excess 488 fluorophore-labeled anti-mouse Fab (Fig. 12.4c). The mouse anti-Ag A labeled with Fab 488 fluorophore is available for use. The excess normal mouse IgG, either free or bound to anti-mouse Fab 488 fluorophore, will not bind to anything during the rest of the experiment. 

It is possible to use HRP as a label for electron microscopic immunocytochemistry because the DAB-developed chromogen generates a reaction product that is easily stained with heavy metals. While HRP can be used for electron microscopic immunocytochemistry, the DAB reaction product does not show high-resolution distribution of the antigen labeled by the 1° antibody. The HRP method has been used to fill specific neurons and trace their dendrites to determine types of synaptic contacts. However, the diffused nature of the reaction product does not allow it to be associated with specific cellular organelles. Therefore, using HRP for electron microscopic immunocytochemistry will not be discussed here. 

Direct immunocytochemistry - a method where the label is bound to the 1° antibody, which then binds to the antigen in the cells. 

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