Immunocytochemistry

Immunocytochemistry (ICC) is a technique for detection and visualization of proteins, or other antigens, in cells using antibodies specifically recognizing the target of interest. The antibody is directly or indirectly linked to a reporter, such as a fluorophore or enzyme. The reporter gives rise to a signal, such as fluorescence or color from an enzymatic reaction, which can be detected in a microscope. The type of microscope used depends on the type of reporter. In ICC, the staining technique is applied on cultured cells or individual cells that have been isolated from eg. tissues, blood samples or mouth swabs. This is in contrast to immunohistochemistry (IHC), where cells are analyzed within intact tissue sections.

Technology

Immunocytochemistry is usually performed in four sequential steps. First, the cells are seeded on a solid support, which is usually a glass slide or a glass-bottom plate. Depending on the type of cells and seeding technique, an incubation time might be necessary before proceeding with immunostaining. In case of seeding adherent cells, the cells will attach to the solid support surface during the incubation, which varies from half an hour to 24 h for the different cell types. In the second step, the cells are subjected to immunostaining, which involves fixation, permeabilization, and antibody incubation. Fixation retains the proteins at their location in the cell and preserves their chemical and structural state at the time of fixation. It can be done by crosslinking or by precipitating the proteins using organic solvents. Upon permeabilization, membranes are punctured with the use of solvents or detergents, allowing the relatively large antibodies to cross the cellular membranes. The permeabilization requires fixation, and hence limits the technique to studying dead cells. During antibody incubation, the antibodies are allowed to bind to target antigens within the cells, after which unbound antibodies are removed by washing. In the third step, the cells and the locations of antibodies bound to target antigens are visualized using microscopy. Images are acquired using a camera or other detector, and in the final step, the images are analyzed and cellular structures annotated. Figure 1 describes a typical workflow for ICC using a fluorescent reporter.


Figure 1. The four steps of immunocytochemistry: (i) cell seeding, (ii) immunostaining, (iii) imaging, and (iv) image analysis.

Reporters

As for IHC, there are different reporter systems available for ICC. One is the use of enzyme-coupled antibodies. After the addition of a substrate, the enzyme catalyzes a reaction that generates a coloured product at the site where the enzyme-coupled antibody is bound in the cells. For example, the commonly used enzyme horseradish peroxidase (HRP) can convert 3,3'-diaminobenzidine (DAB) into a brown precipitate, which can be detected using light-microscopy. Another type of reporter is fluorophores. These molecules can be transiently excited to a higher energy state upon absorption of light with a particular wavelength, and thereafter relax to the ground state while emitting light of a longer wavelength. In this case, a fluorescence microscope is used to excite the fluorophores as well as detecting their emission. Since different fluorophores are excited by different wavelengths of light and also emit light at different wavelengths, multiple fluorophores with different colors may be combined in the same sample. This enables the acquisition of multicolor images, where each color represents a specific antigen target. However, the number of fluorophores used in the same sample is limited by the spectral overlap of the excitation and emission profiles of the fluorophores, as the signals from fluorophores with similar spectral properties cannot readily be separated. In addition to fluorophore-labeled antibodies, there are molecules that are fluorescent by themselves and have intrinsic ability to bind specifically to other molecules. These molecules may be used together with the fluorophore-labeled antibodies. One example is 4',6-diamidino-2-phenylindole (DAPI), which binds to DNA and is commonly used to visualize the cell nucleus. DAPI is excited by ultraviolet light and then emits light in the blue spectrum. A consideration when using fluorophores as reporters is that bleaching will occur when the fluorophores are exposed to light. Over time, the stained sample will decrease in brightness.

Table 1. Examples of different reporters

Reporter type Reporter example Visualization Specificity
Enzyme-coupled antibody Antibody-peroxidase + DAB Brown color Antigen
Fluorophore-labeled antibody Antibody-Cy3 Green excitation / yellow emission Antigen
Biospecific small molecule dye DAPI UV excitation / blue emission DNA

Direct vs. indirect detection

The detection method for the immunostaining can be either direct or indirect. In the direct method, the molecule of interest is directly targeted by a primary antibody linked to the reporter, giving a rapid and specific method. However, it is usually not sensitive enough for most proteins as the number of present copies of the protein is too low to yield a strong enough signal. In the indirect method, the molecule of interest is targeted by an un-labelled primary antibody, which is in turn detected using a reporter-coupled secondary antibody that recognizes the primary antibody (see Figure 1ii). The indirect method is more sensitive due to binding of multiple secondary antibodies to each primary antibody, resulting in signal amplification. Another advantage is also an increased flexibility because of the possibility to vary the primary and secondary antibody combination. Also, since the secondary antibody is targeting the constant region of the primary antibody, which is species-specific, the same secondary antibody can be used for all primary antibodies raised in a given species. The disadvantages of the indirect method are a more laborious and time-consuming protocol, and a risk of non-specific binding of the secondary antibody.

Specific examples

In the Human Protein Atlas, ICC with fluorescence as a reporter (ICC-IF) is used to analyze the subcellular distribution of proteins and build a Cell Atlas of the whole human proteome (Barbe L et al. (2008)). For each protein the subcellular localization is studied in three different human cell lines, mainly using antibodies produced within the Human Protein Atlas project. Cells cultured in vitro, are fixed with paraformaldehyde, permeabilized by treatment with the detergent Triton X-100, and stained by indirect immunofluorescence (Stadler C et al. (2010)). In addition to the antibody targeting the protein of interest, two reference marker antibodies are used to stain the endoplasmic reticulum and microtubules, respectively. and the cells are also counterstained with the nuclear probe DAPI. A confocal laser scanning microscope equipped with a 63x magnification oil immersion objective is used to acquire high-resolution images of the stainings. The images are manually annotated to provide a description of subcellular localization, staining characteristics, and staining intensity. Furthermore, each location is given a reliability score in order to indicate if the results are supported by external experimental data or internal antibody validation. In the end, a knowledge-based revision of the subcellular distribution is performed in a gene-centric manner, taking into account the staining of one or multiple antibodies. Figure 2 shows typical results from ICC-IF in the Cell Atlas.

Figure 2a. RNA binding motif protein 25 (RBM25) localized to nuclear speckles (green). Microtubules are stained in red.


Figure 2b. Golgin B1 (GOLGB1) localized to the Golgi apparatus (green). Microtubules are stained in red and the nucleus in blue (DAPI).


Figure 2c. Electron-transfer-flavoprotein, alpha polypeptide (ETFA) localized to mitochondria (green). Microtubules are stained in red, nucleus in blue (DAPI).


In addition to ICC-IFs, the Human Protein Atlas project has also analyzed protein expression patterns in a panel of cell lines using cell microarrays (Andersson et al., 2006). For preparation of the microarrays, the cells of the different cell lines were fixed in formaldehyde, dispersed in agarose, embedded in paraffin and placed on glass slides. Analysis of protein expression was carried out using Human Protein Atlas generated primary antibodies, HRP-coupled secondary antibodies, and DAB substrate, gresulting in a brown precipitate that correlates with the protein expression. The cells were also counterstained with hematoxylin to give a general staining of the cell structure.

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Applications of Immunocytochemistry - An open-access book about ICC: Ana L. De Paul, J. H. M. J. P. P. S. G. A. A. Q. C. A. M. and A. I. T. Applications of Immunocytochemistry; Dehghani, H., Ed.; InTech, 2012. http://www.intechopen.com/books/applications-of-immunocytochemistry

Immunocytochemistry, a technique for the visualization of proteins and peptides in cells: http://en.wikipedia.org/wiki/Immunocytochemistry

Immunostaining, the use of an antibody-based method to detect a specific target in a sample: http://en.wikipedia.org/wiki/Immunostaining

Immunofluorescence, one type of immunostaining that uses a fluorophore coupled to an antibody for detection: http://en.wikipedia.org/wiki/Immunofluorescence

IHC world – Protocols, Forum, Products, and more: http://www.ihcworld.com/immunocytochemistry.htm

Current Protocols - a continuously updating reference for researchers: http://www.currentprotocols.com/WileyCDA/

The Protocol Exchange - an Open Repository for the deposition and sharing of protocols for scientific research: http://www.nature.com/protocolexchange/protocols

Antibodypedia - An open-access database of publicly available antibodies and their usefulness in various applications: http://www.antibodypedia.com