Immunohistochemistry PDF

Immunohistochemistry Dr. Rakesh Kumar Verma Immunohistochemistry Immunohistochemistry is a technique for identifying cellular or tissue constituents (antigens) using antigen-antibody interactions, the site of binding can be identified by direct label l ing of the antibody or by use of a secon...

Immunohistochemistry Dr. Rakesh Kumar Verma Immunohistochemistry Immunohistochemistry is a technique for identifying cellular or tissue constituents (antigens) using antigen-antibody interactions, the site of binding can be identified by direct label l ing of the antibody or by use of a secondary labelling method. IHC employs a combination of histology, anatomy, immunology and biochemistry to detect the amount, distribution and localization of a specific target within a tissue. The antibodies against the molecule of interest, often a protein, are generated in an organism of a different species and are typically labelled or aided by another set of labelled antibodies. Immunostaining is an umbrella term that encompasses all the techniques that are used for the detection of molecules employing the antigen - antibody reaction. Immunohistochemistry or immunohistochemical staining is a specific use case of immunostaining when the antigen-antibody reaction is used to study the status of molecules in tissue. What’s the difference between immunohistochemistry vs immunofluorescence? The terms immunohistochemistry and immunofluorescence are applied based on the sample type and method of detection used in the immunostaining technique. Immunohistochemistry refers to the evaluation of target antigens in tissues when the detection method can be either chromogenic or fluorogenic. Immunofluorescence, on the other hand, may refer to both the evaluation of target antigens in cells and tissues and specifically involves the detection of a fluorescent label (fluorophore). Key Components Tissue sample Primary antibody (specific to the antigen of interest) Secondary antibody (binds to primary antibody) Detection system (e.g., enzyme, fluorophore) Substrate (for enzymatic detection methods) Amplification Methods Imaging Methods  Tissue The source of the tissue, i.e., the species of the organism of study and the organ of interest, will determine how the tissue is harvested and prepared for slicing. Before performing the experiments, it is vital to refer to literature about the specific organism to understand the techniques involved in harvesting the tissue and the special care that must be taken when performing IHC. Further, it is important to note that before fixation, all tissue must be handled in cold conditions and quickly to prevent rapid decay and drying. Target: The levels and the subcellular localization of the target are extremely important in IHC experiment design. For example, a more abundantly expressed protein can be detected with less effort and a primary antibody tagged with a fluorophore can be sufficient for its detection. However, the detection of less abundant targets would require signal amplification methods. The localization of the target within the cell directly influences the degree of permeabilization required. Therefore, while a nuclear protein would require harsher surfactant-based permeabilization treatment, a cytoplasmic component may be detected using a comparatively milder treatment. Detection of intracellular membrane proteins may be achieved by freeze- thawing alone. Epitope: The epitope is the small three-dimensional surface region of the antigen to which an antibody would specifically bind. The epitope to which the antibody would bind must be exposed at the time of antibody addition during IHC. The epitope that may be recognized by the antibody can sometimes become masked during tissue processing and steps, such as antigen retrieval, may be necessary to expose the epitope.  Fixation methods: Fixation refers to the preservation of the tissue morphology and cell structure in a stationary state by immobilization of the target. It prevents tissue degeneration and enables long- term storage. Typically, soon after harvesting, the tissue is immersed in an appropriate fixative for several hours before it is further processed for sectioning. To minimize the time between tissue harvesting and fixation and to achieve uniform fixation, whole animal transcardial perfusion with the fixative is the preferred method of fixation in animal models, such as rodents.  Fixatives: The choice of fixative and duration of fixation depends on the tissue and the antigen of interest and requires optimization. While insufficient fixation might lead to damaged tissue morphology, prolonged fixation can lead to masking of antigens. Fixatives fall into three categories: aldehydes (formaldehyde and glutaraldehyde), alcohols (methanol and ethanol) and acetone-based fixatives. The most commonly used fixative is a 4% (w/v) paraformaldehyde solution prepared in phosphate buffered saline (PBS).  Sample preparation methods: Sample preparation involves embedding the fixed tissue chunk in a matrix that will enable the tissue to be sliced into sections of even thickness. This step needs to be performed carefully to ensure that the tissue is properly aligned in the matrix of choice. Two common methods of sample preparation are formalin-fixed paraffin embedding (FFPE) and freezing. FFPE involves the dehydration of a tissue sample before gradually embedding it in paraffin wax. Freezing involves cryopreservation of the sample using sucrose before embedding it in an optimal cutting temperature (OCT) compound. Once the tissue pieces are paraffin embedded or frozen, they can be stored under appropriate conditions for longer durations. Sectioning methods: IHC is performed on thin sections of the tissue. The thickness and uniformity of the sections are very important to ensure efficient penetration of the antibody and proper imaging of the tissue. FFPE tissue is commonly sectioned under a microtome at room temperature, whereas frozen tissue is sectioned under a cryostat at sub-zero temperatures. Sections may be obtained along the coronal, sagittal or lateral plane of the tissue. The sections are then mounted on positively charged slides that ensure immobilization of the sections throughout the IHC process. Pre-processing of tissue sections: Once the tissue sections are ready, they can be processed for penetration of the antibody to facilitate the immunogenic reaction. While the FFPE sections need to be deparaffinized using an organic solvent, such as xylene, followed by a rehydration step, OCT compound embedded sections can be used directly for downstream processing. As frozen sections undergo fewer processing steps, this method is more sensitive for the detection of proteins. However, FFPE affords good morphological preservation and helps achieve thinner sections (as thin as 2 microns). Antigen retrieval methods: Formaldehyde-based fixation can often lead to masking of the antigen epitopes. Antigen retrieval is therefore required to unmask the epitopes and make them available for antibody binding and is often performed. This step is important when performing IHC for FFPE tissue, but can be too harsh for frozen tissue sections and may be omitted. Antigen retrieval can be achieved either by the application of heat (heat induced epitope retrieval: HIER) or through enzymatic degradation (proteolytic- induced epitope retrieval: PIER) in an appropriate buffer.  Permeabilization: Permeabilization is an essential first step that renders the plasma membrane of the cells in the tissue porous, thus allowing the entry of IHC reagents and antibodies. Routinely, surfactants such as Triton X-100, Tween- 20, saponin and digitonin are used to achieve permeabilization. However, for a gentler permeabilization for the preservation of intracellular membranes, the sections may be subjected to the freeze-thaw process. Fixatives, such as methanol and acetone, also permeabilize the tissue, and when using these fixatives this step may be omitted. The concentration of the surfactant and the time of incubation are determined based on factors such as the fixative used, the thickness of the tissue section and the subcellular localization of the antigen of interest. Blocking buffer: Although antibody–antigen binding can be very specific, some antibodies may adhere to certain non-specific cellular components due to various intramolecular forces at play. Incubation in blocking buffer before addition of the antibody helps to prevent the non- specific binding of the antibodies in the tissue. Commonly used blocking agents are normal serum and bovine serum albumin. When using chromogenic detection, blocking of endogenous enzyme activity is also required and can be achieved by using hydrogen peroxide to block endogenous peroxidase activity or levamisole to block endogenous alkaline phosphatase activity. Detection method: The detection of the target antigen may be direct, where the label is directly attached to the primary antibody, or indirect, where the label or an enzyme that catalyzes a chromogenic reaction is attached to a secondary antibody. Further, signal amplification methods may be applied to enhance the sensitivity of signal detection. Primary antibodies: The antibody that directly binds to the epitope of the antigen of interest is called the primary antibody. Primary antibody selection is an important step in IHC experimental design and one needs to ensure that the selected antibody is specific to the species under study. The specificity of the antibody against the target antigen also needs to be thoroughly evaluated. Primary antibodies may be polyclonal or monoclonal: while polyclonal antibodies consist of multiple individual antibody molecules that can recognize different epitopes of the same target, monoclonal antibodies all recognize the same single epitope. The concentration of the primary antibody needs to be determined through careful experimentation to achieve the best results. Secondary antibodies: The secondary antibodies are antibodies that recognize the primary antibody and thus enable detection of the target antigen. Secondary antibodies are often tagged with an enzyme to facilitate signal amplification for chromogenic detection, or they may be tagged with a fluorophore. Signal amplification: For target antigens that are expressed at low levels, signal amplification may need to be performed to improve the sensitivity of the technique. Several signal amplification strategies that are used include the avidin-biotin complex (ABC) method, labelled streptavidin-biotin (LSAB) method and tyramide signal amplification (TSA). Label Labels used to detect the target antibody are attached to the primary or secondary antibodies and may be either fluorogenic (as in the case of immunofluorescence) or chromogenic. Fluorogenic labels, such as fluorescein and tetramethylrhodamine (TAMRA), can be directly viewed under a fluorescence microscope. Chromogenic methods involve the conversion of a chromogenic substrate, such as 3,3’-diaminobenzidine (DAB) and 5-bromo-4-chloro-3-indolyl- phosphate (BCIP)/nitro blue tetrazolium (NBT), to a colored product in the presence of an antibody-conjugated enzyme such as, horseradish peroxidase (HRP) or alkaline phosphatase (AP). Mounting: The prepared tissue sections are mounted in a medium with an appropriate refractive index that facilitates imaging under a microscope, protects the fluorescently labeled sections from photobleaching and prevents the section from drying. Commonly used mounting media include DPX, synthetic resins and glycerol-based mounting media containing antifade agents for fluorescently labeled sections. Imaging method: Chromogenic labels can be detected using a light microscope. Fluorescence or confocal microscopes are used for the detection of fluorophores. Electron microscopy may be used for imaging after immunohistochemical labeling with colloidal gold particles  Strengths Limitations Affordable and simple Specificity of antibodies can be procedure that can be variable and needs to be performed with few resources thoroughly checked using appropriate controls Powerful technique to study The method is semi-quantitative, localization and and the absolute abundance of the presence/absence of a target at target cannot be reliably the tissue and cellular level determined Paraffin embedded and frozen Tissue is highly processed and may tissue samples can be stored lead to loss of information of the and accessed when required natural state Stained tissue sections can be IHC is a multi-step procedure and stored and referred to variability can be introduced at any whenever required stage leading to poor reproducibility of results16 What is immunohistochemistr y used for?  IHC is a very popular tool for detection of biomarkers in cancer diagnosis as well as for the development of new biomarkers. Biomarker IHC allows tumor detection, staging and assessme classification, in addition to predicting tumor prognosis and understanding the nt in response of a tumour to treatment paradigms. oncology: IHC-based biomarkers have become vital for the diagnosis and treatment of breast cancer, prostate cancer, pancreatic cancer, lung cancer, bladder cancer, colorectal cancer and ovarian cancer.  IHC serves as an important tool for the detection and identification of pathogenic antigens in tissue samples from infected individuals, which can help in Diagnos the treatment of infectious diseases. es of infectiou s Bacterial pathogens such as, Bartonella quintana, Yersinia pestis, Treponema pallidum, Chlamydia trachomatis; viral pathogens diseases such as, human herpesvirus type 8 (HHV8), Epstein- Barr virus (EBV), human immunodeficiency virus : (HIV); fungal pathogens such as, Candida albicans and Cryptococcus neoformans var. gattii; and protozoal pathogens such as, Plasmodium falciparum and Trypanosoma cruzi have been successfully identified using IHC. Evaluating neurodegenerative disorders: Abnormal protein conformations and aggregations can be identified and evaluated using IHC and are a routine feature of many neurodegenerative disorders including, Alzheimer’s disease (AD), chronic traumatic encephalopathy (CTE), progressive supranuclear palsy (PSP), Pick’s disease, Lewy body disease, multiple system atrophy (MSA) and amyotrophic lateral sclerosis (ALS). Human Protein Atlas: IHC has contributed to the age of big data by enabling the mapping of the human proteome. The Human Protein Atlas is a freely available and valuable resource that contains tissue level information of expression encompassing 90% of all protein encoding genes.