Immunocytochemistry Advantages

Immunocytochemical methods have been widely applied to visualize proteins, carbohydrates, or lipids in sectioned material. The advantage of using immunocytochemistry is to be able to localize the molecules of interest within the tissue. Several procedures have been described. Basically, these procedures can be split into four main steps that are described in subheadings (1) tissue preparation, (2) the primary antibodies, (3) the visualization of the target, and (4) enhancement of signals with antibody complexes. In addition, a protocol for alkaline phosphatase will be presented in detail in Subheading 5. The terms primary and secondary antibodies refer to the order in which they are applied to the target. The immunocytochemical procedures are not limited to sectioned... 

Raman spectroscopy can offer a number of advantages over traditional cell or tissue analysis techniques used in the field of TE (Table 18.1). Commonly used analytical techniques in TE include the determination of a specific enzyme activity (e.g. lactate dehydrogenase, alkaline phosphatase), the expression of genes (e.g. real-time reverse transcriptase polymerase chain reaction) or proteins (e.g. immunohistochemistry, immunocytochemistry, flow cytometry) relevant to cell behaviour and tissue formation. These techniques require invasive processing steps (enzyme treatment, chemical fixation and/or the use of colorimetric or fluorescent labels) which consequently render these techniques unsuitable for studying live cell culture systems in vitro. Raman spectroscopy can, however, be performed directly on cells/tissue constructs without labels, contrast agents or other sample preparation techniques. 

To look at how immunocytochemistry (an individual study) has advantages over an enzyme assay (a population study), let us compare the types of results from these two approaches. To determine the enzyme level, the liver is ground up and a specific biochemical enzyme assay is performed (Fig. 1.1a). At different time points, the level of enzyme activity increases significantly as seen by the small errors shown on the graph (Fig. 1.1a). However, it is easy to assume that all cells in the liver have the enzyme (Fig. 1.1b) detected in the biochemical assay. In reality, however, the... 

Immunocytochemistry takes advantage of three properties of antibodies ... 

Immunocytochemistry labels provide a dizzying list of options. Below are the most important advantages (+) and disadvantages (-) of these two methods. 

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°. 

Electron microscopic immunocytochemistry takes advantage of the increased resolution of the electron microscope to localize label to specific cellular organelles. Optical microscopes limit resolution to the wavelength of light, about 0.2 p,m or 200 nm. The electron microscope has theoretical resolution of less than 0.2 nm. Practically for biological samples, resolution is limited to about 2 nm because of the... 

Quantum dots - heavy-metal nanocrystals that fluoresce when exposed to UV light, a major advantage of Qdots is that they do not photobleach however, their large size makes them difficult to use for immunocytochemistry. 

The general strategies in performing immunocytochemistry of amino acids are essentially the same as those for any other molecule. Immunolabelmg may be performed on sections of embedded tissues, on frozen or Vibratome sections, and on wholemounts, with each approach having its own advantages and disadvantages. 

NADPH diaphorase histochemistry is now the most widely used method for revealing the distribution of NOS in the nervous system. Using this approach, the anatomy of putative NO-producing neurons has been described in the nervous systems of several mammalian species, a variety of other vertebrate species, and invertebrates (7—11). A major advantage of the NADPH diaphorase method over such alternatives as NOS immunocytochemistry and in situ hybridization is that the only specific reagents required are NBT and NADPH, both of which can be easily purchased Moreover, to date anti-NOS antibodies have been raised only to forms of the enzyme obtained from mammals and... 

The significant advantages of the described protocol rest in the versatility of probe design, the increased tissue permeability and hybridization efficiency because of the small probe size and, finally, the superior tissue preservation as a result of the rapid freezmg/dehydration and vapor-induced fixation. In addition, tissue preparation with the proposed protocol is compatible with immu-nocytochemistry. It is possible, therefore, to obtain serial consecutive paraffin sections from the same CNS and to combine in situ hybridization with immunocytochemistry for direct comparison of the signal from the two techniques in the same cells. For a more comprehensive and detailed review of in situ hybridization m general, the reader is referred to the excellent recent practical guide by Leitch et eil.(l) and also to the seminar series by Harris and Wilkinson (2). 

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