Immunostaining uses antibodies to detect the distribution and localization of the specific antigen of interest. IHC markers specific to disease conditions are chosen based on the tissue type and clinical relevance, along with proper control tissues. Clinical information of the antigen like its function, subcellular localization, and expression profile helps in antibody selection. The clonality of the antibodies, polyclonal vs monoclonal, is determined based on the assay specificity.
Polyclonal antibodies recognize multiple epitopes of the same antigen, enhancing the signal. Polyclonal antibodies are more resistant to conformational changes and are used in assays that requires high sensitivity. Monoclonal antibodies are used for high specificity assays as they bind to a single epitope, exhibit low cross-reactivity, and minimal background. BioGenex antibodies come in different formats and volumes to accommodate manual ready-to-use dropper bottles and automated barcoded vials staining procedures.
Our antibodies also come in the concentrated format along with the Enhanced Antibody Diluent for dilution of all antibodies to improve antigen-antibody interaction and affinity and to reduce antibody loss due to adsorption. Primary antibody detection within an IHC experiment can be done using either direct or indirect assays.
In a direct assay, the primary antibody is covalently tagged to a label to detect highly expressed antigens. This method is commonly used for multiplexing with multiple antigen targets. Indirect detection relies on the utilization of a labeled secondary antibody containing reporting enzymes for signal amplification. The target antigen for both methods is visualized using a fluorophore or chromogenic substance. BioGenex Detection Systems are high sensitive kits that give a high-resolution visualization of the bound antibodies with a variety of colorimetric endpoints.
Horseradish peroxide linked to a polymer chain enhances the signal by catalyzing higher molar ratios of the substrates giving intense stains. The kit is non-biotin based to eliminate the cross-reactivity with endogenous biotin to give clean background. This uses a species-specific secondary antibody conjugated to biotin. The bound secondary antibody then reacts with the streptavidin conjugated with an enzyme which on reaction with a suitable substrate forms a colored reaction. Antigen retrieval in formalin-fixed, paraffin-embedded tissues: an enhancement method for immunohistochemical staining based on microwave oven heating of tissue sections.
J Histochem Cytochem.
Immunocytochemistry and Related Techniques | SpringerLink
Detection methods are usually colorimetric or chemiluminescence based. Electron microscopy or EM can be used to study the detailed microarchitecture of tissues or cells. Immuno-EM allows the detection of specific proteins in ultrathin tissue sections.
Antibodies labelled with heavy metal particles e. While powerful in detecting the sub-cellular localisation of a protein, immuno-EM can be technically challenging, expensive, and require rigorous optimisation of tissue fixation and processing methods.
Protein biotinylation in vivo was proposed to alleviate the problems caused by frequent incompatibility of antibody staining with fixation protocols that better preserve cell morphology. In immunostaining methods, an antibody is used to detect a specific protein epitope. These antibodies can be monoclonal or polyclonal. Detection of this first or primary antibody can be accomplished in multiple ways.
The biotin-streptavidin is one commonly used high affinity interaction. As previously described, enzymes such as horseradish peroxidase or alkaline phosphatase are commonly used to catalyse reactions that give a coloured or chemiluminescent product. Fluorescent molecules can be visualised using fluorescence microscopy or confocal microscopy.
The applications of immunostaining are numerous, but are most typically used in clinical diagnostics and laboratory research. Clinically, IHC is used in histopathology for the diagnosis of specific types of cancers based on molecular markers. In laboratory science, immunostaining can be used for a variety of applications based on investigating the presence or absence of a protein, its tissue distribution, its sub-cellular localisation, and of changes in protein expression or degradation.
Immunostaining Immunostaining is a general term in biochemistry that applies to any use of an antibody-based method to detect a specific protein in a sample. Immunostaining techniques Immunohistochemistry Immunohistochemistry or IHC staining of tissue sections or immunocytochemistry, which is the staining of cells , is perhaps the most commonly applied immunostaining technique. Flow cytometry A flow cytometer can be used for the direct analysis of cells expressing one or more specific proteins. This makes the comparison from cell to cell difficult as well as from slide to slide between different tissues and between slides prepared on different days.
This difficulty tends to decrease with the introduction of automatic techniques of coloration. The reagents employed in the immunohistochemistry technique present the potential to give true quantitative results. Most researchers, however, do not consider this possibility because they often do not observe the fact that this technique is no more than an immunological test carried out in situ or in histological slices.
The obtained staining by the immunohistochemistry technique is analogous to the results obtained in an ELISA test enzyme-linked immunosorbent assay , a method recognized worldwide as truly quantitative. Exactly the same reagents that are applied in a serum test of ELISA can be employed for immunohistochemistry reactions in histological slices of specimens in paraffin blocks. As the need for an accurate immunostaining measurement is rising, quantitative biochemical methods of tissue detection are being progressively substituted by immunohistochemistry.
However, advances in molecular biology and the emergence of new treatments for cancer will certainly increase the demand for precise results of a series of new molecules or target-genes, as a patient selection method for a given treatment. Many semi-quantitative measurement methods of immunohistochemistry reactions based on visual scores have been proposed in an attempt to improve this quantification. As seen previously, the tissue expression of biomarkers employed in the immunohistochemistry technique can occur in different cellular compartments and even in extra-cellular matrix constituents.
When the intensity is the evaluation focus, the inclusion of reaction controls containing different levels of staining are required for comparing criteria. Examples of the employment of this methodology includes the count of micro-metastases in bone marrow samples or the measurement of peritumoral blood vessels.
In these examples minimal variances of immunoreactivity intensity, attributed to the staining method or to the fixation procedure, have little impact on the quantification itself. A similar attempt of quantification can be exemplified by the estimate of the proliferation index through immunostaining by Ki67 MIB-1 , or by factors related to the cellular cycle such as P53 and P21, in which a simple count leads to the quantity of normal and neoplastic cells.
Such methods demonstrate low reproducibility and consistency of results in terms of cut off values with relevant sensitivity. Score systems were introduced to clinical practice in an attempt to overcome variances, particularly for markers that aim to select patients for specific treatments. It is important to emphasize that all scores, including those mainly used in daily practice, have demonstrated statistically significant relevance with regards to clinical variables when used by experienced researchers in the area, although they are laborious and fatiguing.
The computer-assisted image analysis has been in use since the s, 97 , 98 without a well defined historical sequence, and has proved superior to the semiquantitative method, especially in terms of its quantification accuracy in many kinds of markers, 82 , 89 , 99 — representing the solution for the reproducibility and applicability of the semi-quantitative score systems, because it yields itself to the desired quantitative result.
The future perspectives point to new discoveries that should make the immunostaining methods simpler. An already available example was the introduction of the reactions performed in a single stage EPOS. Other important achievements were the development of semi-automatic machines especially devised for the immunohistochemistry technique and the microarray technology that will be fundamental in the selection of proteins implicated in diagnosis, prognosis and therapeutic decisions of many diseases.
In addition it could also be of great value in the standardization of the employed technique and the reproducibility of the results. Without doubt, the development of quantification methods for the immunohistochemistry technique, mainly those which are computer-assisted, have increased not only the accuracy in the detection of markers, but also the reliability of their results.
Most larger laboratories, until recently, were those which held this technology compared to small laboratories and academic centers, largely due to economic reasons. At present, immunohistochemistry quantification is widely employed in many areas, not only in pathology, but also in various medical areas with particular impact in the clinical daily practice.
Future developments of the immunohistochemistry technique and its expression quantification should not be disseminated in two languages—that of the pathologist and that of the clinician or surgeon. In order to achieve this goal the collaboration and pooling of knowledge between these two valuable medical areas is vital. National Center for Biotechnology Information , U.
Journal List Biomark Insights v. Biomark Insights. Author information Copyright and License information Disclaimer. Email: rb. This article has been cited by other articles in PMC. Abstract The immunohistochemistry technique is used in the search for cell or tissue antigens that range from amino acids and proteins to infectious agents and specific cellular populations. Keywords: immunohistochemistry, review, pathology. The Immunohistochemistry Technique The immunohistochemistry technique is used in the search for cell or tissue antigens ranging from amino acids and proteins to infectious agents and specific cellular populations.
Open in a separate window. Figure 1. Applications and importance The immunohistochemical reactions can be used in different situations within research or pathological anatomy laboratories. Figure 2. Figure 3. Limitations, difficulties and problems Although a relatively simple technique, immunohistochemistry has some particularities and its outcome depends on many factors.
Interpretation of Immunohistochemistry Expression The interpretation of immunohistochemistry expression is generally made in a qualitative and subjective manner, whereas quantification is considered of little or no importance. Quantification of Immunohistochemistry Expression Soon after the introduction of immunohistochemistry as a routine technique in pathology laboratories, efforts were made in order to try quantify protein expression using immunohistochemistry. Computer-assisted quantitative analysis The computer-assisted image analysis has been in use since the s, 97 , 98 without a well defined historical sequence, and has proved superior to the semiquantitative method, especially in terms of its quantification accuracy in many kinds of markers, 82 , 89 , 99 — representing the solution for the reproducibility and applicability of the semi-quantitative score systems, because it yields itself to the desired quantitative result.
Perspectives The future perspectives point to new discoveries that should make the immunostaining methods simpler. Footnotes Disclosures The authors report no conflicts of interest. References 1. Brandtzaeg P. The increasing power of immunohistochemistry and immunocytochemistry. J Immunol Methods. Immunohistochemistry: forging the links between immunology and pathology. Vet Immunol Immunopathol. Marrack J. Nature of antibodies.
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The unlabeled antibody enzyme method of immunohistochemistry: preparation and properties of soluble antigen-antibody complex horseradish peroxidase-antihorseradish peroxidase and its use in identification of spirochetes. Mason DY, Sammons R. Alkaline phosphatase and peroxidase for double immunoenzymatic labelling of cellular constituents. J Clin Pathol. Rapid preparation of peroxidase: anti-peroxidase complexes for immunocytochemical use. Immunoenzymatic labeling of monoclonal antibodies using immune complexes of alkaline phosphatase and monoclonal anti-alkaline phosphatase APAAP complexes J Histochem Cytochem.
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Immunohistochemistry (IHC) & Antigen/Epitope Protein Retrieval - Principle, Technique and Protocol
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