Immunocytochemistry (ICC) is a common laboratory technique that is used to anatomically visualize the localization of a specific protein or antigen in cells by. Immunocytochemistry (ICC) is a technique for the visualization of proteins and peptides in cells using biomolecules capable of binding the protein of interest. There are three concepts which include immunohistochemistry, immunocytochemistry and immunofluorescence. The assay of immunohistochemistry is relatively.
An immunoglobulin G IgG antibody is a single protein made from four peptides joined by disulfide bonds. There is a single constant region white containing the Fc portion and species-specific antigens. The variable region gray contains the Fab portion that binds the epitope portion of the antigen. The short protein found only in the variable region is known as the light chain; the large protein that is part of the constant and variable region is the heavy chain.
The IgG can be digested by the protease enzyme, papain, at the hinge region flexible region of the heavy chain , into an Fc end constant end and a Fab end variable end. The antigen is the molecule used to immunize the animal, and the epitope is one of many portions of the antigen that can generate antibodies. Figure reproduced with permission from Burry The literature contains a variety of different names for controls, and some controls have multiple names Stirling Additional problems with controls for immunocytochemistry are the wide range of methods in which antibodies and labels are used to localize proteins in cells.
Indirect immunocytochemistry with an unlabeled primary antibody and a species-specific labeled secondary antibody is most popular. Immunocytochemistry for multiple primary antibodies needs controls that show each secondary antibody binds to the correct primary antibody. Avidin-biotin complex ABC uses a third incubation after the secondary antibody to add the enzyme horseradish peroxidase HRP and is also very popular because of its high sensitivity and relative ease to perform.
Today, with many new methods of using antibodies in immunocytochemistry, a new approach is needed to classify and use controls. The controls described below are applicable to immunocytochemistry regardless of the labeling method used. Labeling methods used in immunocytochemistry can be grouped in three types: Fluorescence relies on light emitted from a fluorophore with a different wavelength than the light used to excite the fluorophore.
Histological enzymes are proteins that convert an uncolored water-soluble substrate to a colored water-insoluble product, with HRP a commonly used enzyme. Particulate labels are heavy metals such as colloidal gold or small gold particles that are used mainly for electron microscopic immunocytochemistry. To simplify the use and discussion of immunocytochemistry controls, three types of controls were proposed Burry This organization of the controls is based on the function of the individual control and its use in the immunocytochemical procedure.
The new terms for the controls are designed to better describe their function in the immunocytochemical procedure. With a better understanding of the function of the controls and the new terms, it is hoped that the use of controls will become more consistent. The primary antibody control is a specificity control and confirms that the primary antibody binds to the correct epitope on the expected antigen.
Ideally, this control demonstrates binding specificity under the same conditions of the fixed cells or tissue section. Thus, the primary antibody control is not just showing the antibody is specific for the antigen, but it also shows the effects of fixation and detergent treatment on the tissue or cells.
A broad range of methods have been used to show specificity of the primary antibody for the antigen. The lack of a single widely accepted method for determining the specificity of the primary antibody confirms the difficulty of demonstrating this point.
Four methods are discussed as primary antibody controls. The best primary antibody control is to use a genetic approach to manipulate the expression of the antigen protein. Within the genetic approaches, the first method is to use tissue from a knockout animal Saper and Sawchenko This approach removes the protein and allows the tissue to be prepared in the same manner as the experimental sample.
With a nonfunctional gene, the protein of interest is not expressed, and the primary antibody should not bind to the tissue. Tissue from these animals can be fixed and prepared using the same procedure as the experimental animals to minimize experimental conditions affecting the result of the control. However, there are problems with knockout as a primary antibody control. Not only are knockout animals for specific proteins not common, but even if one is found, it may be difficult to get access.
Some knockout animals are functional knockouts that still express the protein, but the protein is mutated or non-functional Lorincz and Nusser In these cases, the primary antibody to the protein could still bind to the defective protein in the knockout animal. The second genetic method used for a primary antibody control is the transfected cell line expressing the antigen for the primary antibody Saper This control includes the conditions of fixation and shows the primary antibody works with the method used.
The untransfected cells serve as a negative control because they do not express the protein. Also, using siRNA to knock down the expression of the antigen protein can be used M. Willingham, personal communication, A third genetic method used as a primary antibody control is the combination of immunocytochemistry and fluorescent in situ hybridization Rhodes and Trimmer The concept is that synthesis of a protein from mRNA should be close to the site where the antibody detects the protein.
One drawback to this approach is that the methods for preserving tissue for immunocytochemistry and in situ hybridization are not fully compatible, leading to less than optimal signal.
This problem is combined with the fact that mRNA for some proteins in developing cells is silenced with RNA binding proteins, and the mRNA is transported within the cell before translatiion.
The second primary antibody control method is immunoblot Western blot , a very reliable and most common method of determining the specificity of the primary antibody. In this case, the antibody labels one protein at the correct molecular weight.
These immunoblots are relatively easy to do and thus are the most common form of specificity control seen for commercial antibodies. This approach is relatively inexpensive and straightforward. The problem with immunoblots is that the protein is not fixed but is denatured in SDS, and so they lose their secondary and tertiary structure.
Some antibodies bind only to denatured protein immunoblots, and some antibodies bind only to native proteins in immunocytochemistry Willingham When obtaining an antibody, it is advisable to consider only those that have been tested or used for immunocytochemistry.
A third method for primary antibody controls is colocalization with the original primary antibody and an additional label to show that they both bind to the same structure. In this case, two different primary antibodies to different epitopes on the same antigen frequently to different epitopes, such as the C- and N-termini of the same protein give the same label site to confirm the primary antibody is specific for a protein.
Another way to perform double labeling is to take advantage of a fluorescent protein as part of a specific protein to show double labeling with immunocytochemistry. Here, the transfected cells with a fluorescent protein e. The general problem with colocalization is that although it shows that the labels are in the same spot, it does not show that they bind to the same protein because the resolution of the light microscope is not enough to resolve the location of a single protein.
The fourth method for primary antibody controls is absorption controls. The absorbed antibody can no longer bind to antigens in the section Fig. To perform this control, correctly purified antigen or peptide antigen and not a crude homogenate is critical.
The next step is to show that no unbound primary antibodies are present after the absorption. In a separate experiment, a titration curve is done to determine the concentration of the isolated antigen that saturates the antibody, leaving no unbound primary antibodies.
Absorption control is the incubation of the primary antibody with the antigen used to generate the antibody. A The primary antibody incubated with excess antigens binds all of the Fab sites capable of binding the antigen in the tissue arrow. B If the correct antigen and an incorrect antigen have the same epitope arrows , then binding to both is inhibited by the absorption control. C In some cases, the antigen adsorbed by the antibody binds to proteins in the tissue, and the adsorbed antibody appears to bind to a protein 4 independently of the antibody.
Two major problems with absorption controls limit its usefulness Burry First, if a specific primary antibody binds to the same epitope on the protein of interest and a second protein, absorption by one protein in the absorption incubation inhibits binding of the antibody to all potential proteins Fig. The absorption control does not exclude the binding of the primary antibody to proteins other than the one used for incubation. This would be a false negative, indicating the antibody is specific for one antigen.
Examples of such false negatives occur when the epitopes are shared on several antigens and bind to all antigens by absorption e. To ensure a negative absorption control is correct, an immunoblot must show that the epitope is found on a single protein e. Thus, the absorption control needs an additional control to eliminate the possibility that the primary antibody is bound to multiple antigens. A second problem with absorption controls is that the proteins, even when bound to the primary antibody, can retain the ability to bind other proteins in cells.
The bound protein could then bind to the tissue independent of the antibody and give a false-positive labeling Fig. One solution is to bind the antigen to beads and remove the adsorbed antibody from the solution Storm-Mathisen and Ottersen However, the best absorption control is the use of small peptides for antigens because only the epitope of the antigen is used for absorption.
The secondary antibody control shows that the labeling observed is due only to binding of the secondary antibody to the primary antibody. This control is done by either eliminating the primary antibody or replacing it with the same amount of normal serum from the same species. With no primary antibody to bind the secondary antibody, no labeling should be seen. The secondary antibody control needs to be run in parallel with each experiment.
There are several types of problems detected with this control: A Incubation with antibodies should show antibody binding to only the correct antigen. Nonspecific binding can result from charged groups that bind proteins, including antibodies.
The tissue can have Fc receptors that bind to the Fc region of any antibody. Some tissues can have exposed endogenous antibodies. In experiments with multiple primary antibodies, incorrect binding by other antibodies can occur. B There are blocking agents that block each of the sites that cause nonspecific binding. The primary antibody binds to the correct antigen and is not affected by any of the blocking agents.
Charged groups can be quenched by any protein, and BSA is commonly used because it is not a source of antibody binding. The Fc receptors must be quenched by the Fc end of an IgG antibody that has no ability to bind other antigens; normal serum from the same species as the secondary antibody is commonly used.
Endogenous antibodies are blocked by incubations with antispecies Fab fragments that are used only when tissue from injured animals is processed. In experiments with multiple primary antibodies, where incorrect binding of labeled secondary antibodies is suspected, the secondary antibody control shows this incorrect binding.
Reproduced with permission from Burry Binding of secondary antibodies to charged groups occurs in fixed tissue sections and cell cultures Fig. Charged groups in cells are generally positively charged and bind proteins with negative charged groups. The charged groups are unbound aldehydes from the fixative or cell components such as histones.
The type of sample being tested determines if there needs to be an incubation period before the staining can begin, which type of reporter will be used, and what microscope is needed for visualization. The body is made up of many different types of proteins, peptides, and antigens that help carry out functions of the immune system. Antigens are single molecules that combine to form peptides. Peptides are called proteins when they contain more than fifty amino acids.
The identification of proteins in cells is important in recognizing the body's immune response to various antibodies. Antibodies are used to fight infections in the body. Immunocytochemistry helps researchers determine which antibodies will bind to specific antigens and build immunity.
Phil is now ready to start applying immunocytochemistry to his samples. Some samples require direct detection while others require indirect detection. He gathers the samples that are ready after necessary incubation time. There are four basic steps to complete immunocytochemistry testing. The first step is cell seeding where the samples are placed on glass slides or transparent plates. Samples require different incubation times before they can be used. Some samples are used right away while others must set for several hours or even twenty-four hours.
The next step consists of fixation of the cells and then immunostaining the cells. A fixative solution is applied to the cells so that they will not move on the slide. Some slides also need to be permeabilized with another solution in order for the stain to react depending on the type of fixative used.
The antibody or immunostain reporter is then applied to the slide and later rinsed off to ensure excess antibodies are removed.
The third step uses the microscope to visualize the sample and images are developed. The fourth step involves analyzing the images that are written down as an explanation.
There are different reporters used to identify proteins in the cells. It is important to properly label the slides so information is accurate. The different types of reporters that are used to stain the cells are:. The direct method of immunocytochemistry only uses one antibody for immunostaining, which is done quickly and is precise. However, it may not be sensitive enough because it requires many proteins to be present to show a reaction.
Indirect immunocytochemistry uses a primary and secondary reporter. The primary antibody is applied to the sample and then a secondary reporter-paired antibody is applied to the primary antibody. This process takes longer but increases the sensitivity of the reaction. Phil used multiple methods throughout his workday depending on what sample he was testing. He was sure to label each sample slide correctly and take clear images to send to be analyzed. Immunocytochemistry is a process used in the laboratory setting to visualize antigens , peptides , and proteins in cells.
Different types of antibodies are then applied to the samples of cells using different types of reporters including enzyme-coupled antibodies and fluorescences.
The steps involved in completing immunocytochemistry are seeding where the samples are applied to glass slides, fixation , and immunostaining where samples are secured and stained for visualization, using a microscope to view the samples, and then analyzing the images.
Immunocytochemistry is done using a direct or indirect method. The direct method uses one antibody while the indirect method uses a primary and secondary antibody. The information on this site is for your information only and is not a substitute for professional medical advice.
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To learn more, visit our Earning Credit Page. Not sure what college you want to attend yet? The videos on Study. Explore over 4, video courses. Find a degree that fits your goals. Try it risk-free for 30 days. Add to Add to Add to. Want to watch this again later? Immunocytochemistry is used as a laboratory technique to identify proteins and peptides.
This lesson will discuss the definition, uses, and techniques of immunocytochemistry. Laboratory Testing Phil is working in the laboratory and assigned to complete immunocytochemistry testing. What Is Immunocytochemistry Immunocytochemistry is a procedure that is done in a laboratory that visualizes proteins and peptides. Microscope slide sample for viewing Immunocytochemistry Uses The body is made up of many different types of proteins, peptides, and antigens that help carry out functions of the immune system.
Technique for Immunocytochemistry There are four basic steps to complete immunocytochemistry testing. Microscope used in immunocytochemistry There are different reporters used to identify proteins in the cells. The different types of reporters that are used to stain the cells are: Try it risk-free No obligation, cancel anytime.
Immunohistochemistry (IHC) vs. Immunocytochemistry (ICC)
While the term immunohistochemistry (IHC) is often used interchangeably with immunocytochemistry (ICC), significant differences exist between IHC and ICC in . Immunofluorescence, Immunohistochemistry and Immunocytochemistry are often used interchangeably. They are however clearly distinct, find out the answer to. Immunocytochemistry (ICC). Description: ICC is used to understand the distribution and localization of proteins within compartments of a cell. ICC detects .