Immunocytochemistry (ICC) is a technique for the detection and visualization of proteins and peptides in cells using biomolecules capable of binding the protein of interest. Usually the biomolecule is an antibody that is directly or indirectly linked to a reporter, e.g. a fluorophore, fluorescent dye, or enzyme. The reporter will give rise to a signal, e.g. fluorescence or color from an enzymatic reaction, which is then detectable by a microscope. The type of microscope used for image acquisition depends on the type of reporter. In ICC the staining technique is applied on cultured cells or cells with the extracellular matrix removed, in comparison to immunohistochemistry (IHC), where a mix of different cells within a tissue section is analyzed in situ. The biosample can be a tissue section, mouth swab, blood sample, or any sample taken from an individual, animal, or plant.
Immunocytochemistry is usually performed in four sequential steps. First, the cells are seeded on a solid support, e.g. into a 96 well plate with glass bottom or on a glass slide. Depending on the type of cell and seeding technique, an incubation time might be necessary before proceeding with immunostaining; e.g. in the 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 immunostained: cells are fixed, permeabilized, and stained with antibodies. 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. During permeabilization, membranes are punctated with the use of solvents or detergents, allowing the antibodies to cross the membranes. Without this step the antibodies are restricted to the outside of the cell due to their size. The permeabilization requires fixation and hence limits the technique to studying dead cells. After incubation with antibodies, washing steps are applied to remove any unbound antibodies. In the third step, the cells are visualized using a microscope, and images are acquired using a camera. In the final step, the images are analyzed and cellular structures are 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.
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 enzymes catalyze a visible color reaction at the place where the enzyme-coupled antibody is bound in the sample. For example, the commonly used enzyme horseradish peroxidase (HRP) can convert 3,3'-diaminobenzidine (DAB) into a brown precipitate that is deposited in the sample at the site of the reaction. The brown color stain may be seen using light-microscopy. Another type of reporter is fluorescence, which relies on the physical properties of a molecule to be excited to a higher energy state by absorbing light of one wavelength. Thereafter the molecule relaxes to the ground state, while emitting light of a longer wavelength. These molecules are called fluorophores, and they may be visualized using a fluorescence microscope. This type of microscope is able to excite the fluorophores, while at the same time 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. The spectral overlap of the excitation and emission profiles of the fluorophores is the limiting factor when adding different types of fluorophores to a sample. Fluorophores with similar spectral properties cannot readily be separated and the resulting image will show an indistinguishable mix of different signals, which limits the versatility of the immunofluorescence analysis. 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 DNA. It is excited by ultraviolet light and then emits blue light. 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
Direct vs. indirect detection
The detection method for the immunostaining can be either direct or indirect. In the direct method, one reporter labeled antibody is used; this method is rapid and specific. 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. Targeting a specific protein in a cell with a primary antibody and a reporter-coupled secondary antibody directed at the primary antibody is a much more sensitive method (see Figure 1ii). The increased sensitivity of the indirect method is due to the multiple binding of secondary antibodies to the primary antibody, which amplifies the signal. Another advantage is also an increased flexibility because of the possibility to vary the primary and secondary antibody combination. 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.
In the Human Protein Atlas, ICC with fluorescence as a reporter is used to analyze the subcellular distribution of proteins to build a Cell Atlas with subcellular resolution of the whole human proteome (Barbe et al., 2008). For each protein the subcellular localization is studied in three different human cell lines, using the antibodies produced in the Human Protein Atlas project. The cells are cultured in vitro, fixed and permeabilized by tretment with formaldehyde and Triton X-100 detergent, and then immunofluorescently stained (Stadler et al., 2010). In addition to the Human Protein Atlas antibodies, 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 staining. The images are subsequently manually annotated and the subcellular localization, characteristics, and staining intensity are described. Further, a validation score is set for each antibody indicating if the results are supported by other experimental data. 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 using immunofluorescence.
Figure 2a. RNA binding motif protein 25 (RBM25) localized in the nuclear speckles (green). Microtubules are stained in red.
Figure 2b. Golgin B1 (GOLGB1) localized in the Golgi apparatus (green). Microtubules are stained in red, nucleus in blue (DAPI).
Figure 2c. Electron-transfer-flavoprotein, alpha polypeptide (ETFA) localized in mitochondria (green). Microtubules are stained in red, nucleus in blue (DAPI).
Besides the subcellular immunofluorescence studies, the Human Protein Atlas project has also analyzed the panel of cell lines and their protein expression patterns (Andersson et al., 2006). For the preparation of the microarrays, the cells of the different cell lines are fixed in formaldehyde, dispersed in agarose, embedded in paraffin and then placed on glass slides. The analysis of the protein expression is carried out by using Protein Atlas generated primary antibodies, HRP-coupled secondary antibodies, and DAB substrate. The outcome is a brown precipitate, which correlates with the protein expression. The cells are also counterstained with hematoxylin to give a general staining of the cell structure. In addition, mRNA expression profiles (RNA-Seq) of these 56 cell lines have been generated.
References and Links
Andersson AC et al, 2006. Analysis of protein expression in cell microarrays: a tool for antibody-based proteomics. J Histochem Cytochem.
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