Immunohistochemistry Technique 2024-2025 PDF

IMMUNOHISTCHEMISTRY TECHNIQUE Prof. Dr. Ayhan BİLİR 2024- 2025 What are the principles of immunohistochemistry? Immunohistochemistry is a research method combining morphology and biochemistry. A brief summary of this basic step is to react with "antibody" that specifically binds to an...

IMMUNOHISTCHEMISTRY TECHNIQUE Prof. Dr. Ayhan BİLİR 2024- 2025 What are the principles of immunohistochemistry? Immunohistochemistry is a research method combining morphology and biochemistry. A brief summary of this basic step is to react with "antibody" that specifically binds to an "antigen", Bilir A and "visualize" the site where the antigen-antibody reaction has taken place and observe it microscopically. Bromodeoxyuridine İmmunohistochemical Staining Technique In the laboratory, antibodies can be purified from the blood and conjugated(attached) to a fluorescent dye. In general , fluorescent dyes(fluorochromes are chemicals that absorb light of different waveelegths(eg., green,yellow, red). Fluorescein , the most commonly used dye, absorbed ultraviolet light and emits green light. When is immunohistochemistry performed? An IHC can be used to: Diagnose a condition: An IHC allows to diagnose conditions like cancer. It can help to determine the type of cancer (for example, carcinoma, melanoma or sarcoma). It also allows the origins of cancer that’s spread (metastatic cancer). Determine prognosis: An IHC can determine how high-risk, or aggressive, a cancer is. It also can help providers stage and grade cancer. This information can help to find the best treatment way. Treatment to response: it can help to determine treatment response. Researchers also perform IHC to develop new drug discover and treatments. IHC helps researchers learn more about how the smallest cells work How does the test work? An IHC uses antibodies to detect a target antigen in a tissue sample. The target antigen is a marker, indicating a specific disease is present. If the antibody recognizes the antigen, it will attach (bind) to it. The binding process is similar to a lock (antigen) and key (antibody). If the antibody binds to the antigen, the tissue sample will stain a certain color when viewed beneath a microscope. What are the limitations of immunohistochemistry? There aren’t standard guidelines for each step in immunohistochemistry. Different labs use different techniques, which means results may vary. Also, recent research has shown that not all antibodies available for IHC detect the target antigen in a sample. If there are problems with the antibodies, a test may give results that are false, including: False-positive: An IHC detects an antigen that is not present. False-negative: An IHC doesn’t detect an antigen that is present. Labs must have quality controls in place so that every step preserves the tissue and ensures a high-quality stain. To improve IHC accuracy, researcher can test antibodies on tissue known to contain the target antigen to ensure it stains before testing an unknown tissue sample How accurate is immunohistochemistry? When performed correctly and with quality controls in place, immunohistochemistry is a reliable method for cancer diagnosis. One study reports that IHC can accurately identify the primary location of metastatic cancer with 70% to 90% accuracy. An Introduction to Immunohistochemistry Immunohistochemistry (IHC) is a technique for identifying cellular or tissue antigens by means of antigen-antibody interactions. Target Antigen Antigens are proteins that are within or on the surface of a cell. Pathologists look for the presence or absence of particular antigens to assist with diagnosis. There are many hundreds of antigens that have been found to be diagnostically useful. Antigen : In immunology, an antigen (Ag) is a molecule or molecular structure or any foreign particulate matter or a pollen grain that can bind to a specific antibody. An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identify and neutralize foreign objects.  What Are The Basic Steps Of Immunohistochemistry There Are Four Main Steps A general immunohistochemistry protocol consists of four main steps: 1. Sample Preparation; Fixation—to keep everything in its place. 2. Antigen retrieval— to increase the availability of proteins for detection. 3. Blocking—to minimize pesky background signals. 4. Antibody labeling and 5.Sample Visualization—to get the pretty pictures. Table 1: Methods used to retrieve antigens. Direct and Indirect Methods Direct; Assays are classed as direct if the label is conjugated directly onto the primary antibody. Indirect; Indirect (primary antibody + conjugated secondary antibody ) Figure 1. Indirect staining methodology. Figure 2. Direct staining methodology Human antibodies are classified into five isotypes (IgM, IgD, IgG, IgA, and IgE) according to their H chains, which provide each isotype with distinct characteristics and roles. Primary Antibody The first stage of IHC is the application of a primary antibody that binds specifically to the target antigen. There are two main types of antibody, 1. Polyclonal antibodies have an affinity with, and bind to, multiple epitopes (or parts) or the target antigen,. 2. Monoclonal antibodies have an affinity to only one epitope, This type staining is more specific staining but are less sensitive or intense.An antibody (Ab), also known as an immunoglobulin (Ig), is a large, Y-shaped protein used by the immune system to identi fy and neutralize foreign objects such as pathogenic bacteria and viruses. The antibody recognizes a unique molecule of the pathogen, call ed an antigen. Polyclonal antibodies are made using several different immune cells. They will have the affinity for the same antigen but different epitopes, while monoclonal antibodies are made using identical immune cells that are all clones of a specific parent cell What is the epitope and Paratope An epitope, also known as antigenic determinant, is the part of an antigen that is recognized by the immune system, specifically by antibodies, B cells, or T cells. The part of an antibody that binds to the epitope is called a Paratope Epitopes vs. Paratopes. Epitopes are formed by amino acids on the target antigen, while paratopes are formed by amino acids on the binding antibody. Epitopes and paratopes interact to define the location and kinetics of binding. A monoclonal antibody is an antibody produced from a cell l ine made by cloning a unique white blood cell. All subsequent antibodies derived th is way trace back to a unique parent cell. I request you to pay attention to this statement. Antibodies are glycoproteins belonging to the immunoglobulin superfamily. Some antibodies also form complexes by binding to antigen: this is called an antigen-antibody complex or immune complex. Each B cell, produced in the bone marrow, is highly specific to an antigen B cells create antibodies. B lymphocytes, also called B cells, create a type of protein called an antibody. B cells participate in T-cell activation via antigen presentation, costimulation and cytokine production; What is the general role of B cells in the immune system? B-cells are the type of cells that produce antibodies to fight bacteria and viruses. B cells are at the centre of the adaptive humoral immune system and are responsible for mediating the production of antigen-specific immunoglobulin (Ig) Secondary Antibody Next, secondary antibodies bind to the primary antibody. This is known as indirect IHC. It is now commonly used as multiple secondaries can bind to a single primary to amplify the staining intensity. Detection Systems Detection systems create a complete IHC and ISH staining solution. Kits include polymer detection systems, ancillary reagents and consumables for the entire IHC/ISH process, and have both manual and automated options. The Chromogen Finally, a substrate forms an insoluble colored precipitate that can be visualized under a microscope. There are two commonly used chromogens: 1. DAB (brown. 3,3'-diaminobenzidine). 2. AP (red. alkaline phosphatase). DAB is used for most applications as it provides strong and permanent stains. AP Red (or another red chromogen) is used mainly for skin sections DAB 2.Alkaline Phosphatase (AP) Chromogen - Red DAB HRP Brown Chromogen Typical chromogens in multiplexed IHC assays. IHC, or ImmunoHistoChemistry, is a special staining process performed on fresh or frozen breast cancer tissue removed during biopsy. IHC is used to show whether or not the cancer cells have HER2 receptors and/or hormone receptors on their surface Bilir Bilir A A BrdU Bromodeoxyuridine immunostaining methods. İstanbul Faculty of Medicine Breast cancer tissue Immunohistochemisty stain with BRdU and on the right mitosis Bilir A Bilir A Thank you for listening