priciples and applications Immunohistochemistry

basic introduction, principles and methods of IHC

1. Immunohistochemistry Presentation Markos Tadele Tuesday, April 26, 2016

2. Contents Introduction Principle Method General IHC protocol Immunostaining

3. Introduction Immunohistochemistry ( IHC) Combines histological, immunological and biochemical techniques for the identification of specific tissue components by means of a specific antigen/antibody reaction tagged with a visible label IHC makes it possible to visualize the distribution and localization of specific cellular components with in a cell or tissue.

4. Principle The principle of immunohistochemistry is the localization of antigens in tissue sections by the use of labeled antibodies as specific reagents antigen-antibody interactions that are visualized by a marker such as fluorescent dye, enzyme, radioactive element or colloidal gold.

5. ANTIGENS AND ANTIBODIES Antigens An antigen is a substance foreign to the host which stimulates formation of a specific antibody and which will react with the antibody produced. Antigens have two main properties. immunogenicity, which is the ability to induce antibody formation. specific reactivity, which means that the antigen can react “with the antibody it caused to be produced.

6. Antigen The reaction between an antigen and its antibody is one of the most specific in biology. Antigen-mainly proteins, glycoprotein, polysaccharides Complementary Determining Region

7. Antibodies An antibody is a serum protein that is formed in response to exposure to an antigen. Reacts specifically with that antigen to form immune complexes either in the body or in the laboratory. Antibody production is a response by the body to foreign material (an antigen), and is designed to rid the body of this invader.

8. Antibody Structure Structurally, an antibody is made up of two kinds of protein chains - heavy and light chains. Immunoglobulins are named for their heavy chains IgG molecule have heavy chains of the gamma type. An IgA antibody has alpha heavy chains; IgD , delta heavy chains. IgE , epsilon heavy chains IgM has mu heavy chains. A primary antibody for immunoperoxidase staining that is "specific for gamma chains"will localize the heavy chain of an IgG molecule.

9. Light chains There are only two types of light chains common to all five groups: kappa and lambda. An IgG molecule has two identical light chains. either two kappa chains or two lambda chains A single antibody can never have both kappa and lambda chains.

10. Antibody

11. Antibody production A source for the antigen such as serum , urine or tissue is subjected to a combination of procedures including precipitation, centrifugation, dialysis… Other techniques like chromatography and electrophoresis to obtain a highly purified antigen. The antigen is then injected into an animal, not same species.

12. Antibody production Antibody production begins within twenty minutes after injection, although a measurable quantity of antibody cannot be detected for 5-10 days. Sometimes boosting is important

13. Monocloanal and polyclonal antibodies Polyclonalantibodies Large complex antigens may have multiple epitopes and elicit several antibody types. Mixtures of different antibodies to a single antigen are called polyclonal antibodies. Monoclonalantibodies Antibodies specific for a single epitope and produced by a single clone are called monoclonal antibodies and are commonly raised in mice.

14. Antibody caharacteristics Polyclonal 1. in general produce stronger signals 2. greater potential for false positive staining due to antibodies cross-reacting to undesired targets(affinity purification helps) 3. limited supply

15. Monoclonal 1. highly specific 2. less background 3. intrinsic cross-reactivity to non-target can be problematic 4. potential for epitope loss = loss of staining 5. unlimited supply

16. Polyclonal antibodies are made by injecting an animal with your target antigen . Immune response Monoclonal antibodies are made by injecting an animal with target antigen. spleen cells Myeloma cells HGPRT - fused Monocloanal and polyclonal antibodies

17. Summary Polyclonal antibodies Monoclonal antibodies Inexpensive to produce Expensive to produce Skills required for production are low Training is required for the technology used Relatively quick to produce Hybridomas take a relatively long time to produce​ Generate large amounts of non-specific antibodies Generate large amaounts of specific antibodies Recognize multiple epitopes on any one antigen  Recognize only one epitope on an antigen Can have batch-to-batch variability Once a hybridoma  is made, it is a constant and renewable source

18. Conjugation/ labelling Antibodies are not visible with standard microscopy and must be labeled in amanner that does not interfere with their binding specificity. Conjugation is the process of chemically linking some type of marker onto an antibody molecule. A wid e valriety of conjugates are available for use In various direct and indirect immunohistological stains.

19. Conjugation/ labelling Common labels include: Fluorochromes ( fluorescein , rhodamine ) Enzymes demonstrable via enzyme histochemical techniques ( peroxidase , alkaline phosphatase ) electron scattering compounds for use in electron microscopy ( ferritin , colloidal gold).

20. Step Effect on IHC Biopsy Depending on the appropriate tissue type , tissue samples can be obtained in different ways such as punch/core biopsy, excisional / incisional etc. Tissue degradation begins at the time of sample removal. Fixation The sample should be fixed as soon as possible after sampling, ideally within less than an hour. The chemical fixation crosslink proteins in the sample thereby stopping the degradation process . Embedding After fixation, the sample is embedded in paraffin for long-term storage and to enable sectioning for subsequent staining. Once embedded in paraffin, samples can be stored (almost) indefinitely. Sectioning and Mounting Formalin-fixed, paraffin-embedded tissues are sectioned into thin slices (4-5 μm ) with a microtome. The sections are then mounted onto adhesive-coated glass slides.

21. Antigen Retrieval Due to the fixation process, an antigen retrieval treatment is applied to unmask the epitopes , either by heat (heat-induced epitope retrieval; HIER) or enzymatic degradation ( proteolytic -induced epitope retrieval; PIER). Primary Antibody The specificity and sensitivity of the antibody affect the staining result. Visualization The antigen/antibody complex signal is amplified and visualized using a detection system. The strength of amplification of the reaction affects the staining result (intensity). Interpretation The staining pattern is assessed by a pathologist in context with other biomarkers, controls and other tests (e.g. H&E, special stains.

22. Fixation Goals of fixation Prevent autolysis by rapidly terminating enzymatic/metabolic activities Prevent bacterial decomposition. Preserve tissue structures while stabilizing and hardening the tissue for processing.

23. Slide Preparation IHC-P: Paraffin-embedded Cell Pellets and Tissue Prior to immunostaining , harvested and fixed in 10% neutral buffered formalin (NBF) to preserve cell morphology and target epitopes . IHC-F: Frozen Tissue Frozen tissue should be stored at -80°C When ready to stain, equilibrate tissue at -20°C for 15 minutes before attempting to section. Section the tissue to a 6-8 μm thickness using a microtome

24. Slide Storage freshly cut slides- For best results Slides may lose antigenic potential over time in storage. If slides must be stored, do so unbaked at 4°C.

25. Deparaffinization / rehydration Paraffin wax must be completely removed- for staining This is done through a series of sequential xylene /ethanol/water washes that remove the wax and rehydrate the tissue for subsequent antibody binding. Insufficient paraffin removal can lead to spotty, uneven background staining.

26. Retrieving antigens “lost” during fixation Frozen sections were commonly used to bypass the problem of ”over fixation” but gives poor morphology • Limited protease treatment could allow successful antibody staining in previously negative tissues Cross links can block antibody access to target epitopes . Treatment with protease can re-expose epitopes (“antigen retrieval”)

27. Heat induced antigen retrival Heat treatment could dramatically improve immunostaining results: HIER (heat induced epitope retrival ) Used with different buffers • microwave • heat plate (immersion) • steamer • pressure cooker/autoclave

28. Enzymatic Antigen Retrieval Antigen retrieval can also be achieved through enzymatic digestion with pepsin, trypsin , or proteinase K. For those antibodies that require enzymatic retrieval rather than HIER, the recommended enzyme and digesting conditions will be clearly indicated on the product datasheet.

29. Primary antibody Antibody Incubation overnight primary antibody incubation at 4°C, and all is recommended Washing Adequate washing is critical for contrasting low background and high signal. Wash slides three times for 5 minutes with water or other solvent after antibody incubation.

30. Blocking For IHC-P- recommended blocking the samples in buffered saline plus 5% normal goat serum (NGS) for 1 hour at room Temperature prevent nonspecific background staining. Block samples.

31. Counterstains After antibody detection but before cover slip it is advisable to counter stain the tissue to visualize cellular anatomy and orient the viewer in respect to the specific staining.

32. Detection and immunostaining Avidin biotin-complex (ABC) require a 2-step process of detection involving biotinylated secondary antibody followed by exposure to an avidin-peroxidase complex prior to chromogenic detection. liver and kidney that possess high levels of endogenous biotin, (polymer-based detection systems)

33. detection / Immunostaining fluorescent technique Direct method indirect method

34. Immunoflourescent techniques • Advantages: – Hi-resolution, easy to double/triple label – Better sub cellular detail – Can be used with 3D microscopy/live imaging • Disadvantages: – Background Auto fluorescence – Cost – Lack of surrounding tissue/cellular detail – Not permanent

35. Peroxidase (HRP horse reddish peroxidase ) producing good sensitivity Most commonly used Most often used substrate: 3,3'-Diaminobenzidine Tetrahydrochloride (DAB) DAB reacts with HRP to form a brown precipitate at the site of antibody binding.

36. Other Peroxidase Substrates 4-chloro-1-naphthol = BLUE 4-napthol pyronin = RED-PURPLE 2,2’-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid) (ABTS) 3-amino-9-ethylcarbazole (AEC) = RED VECTOR Nova = RED VECTOR VIP = PURPLE

37. Enzyme linked immunostaining Antibodies cross-linked to enzymes that generate an intensely colored Alkaline phosphatase 5-bromo-4-chloro-3-indolyl phosphate/ nitroblue tetrazolium (NBT) – Vector Red – Vector Blue – Vector Black etc • Glucose oxidase – with nitroblue tetrazolium • B-D- Galactosidase

38. Mounting and examination Aqueous and non aqueous (permanent) mounting media are available. The mounting media depends on the chromogen used during the detection step and its solubility in organic solvents or water Water insoluble Chromogens should not be used with nonaqueous mounting media andwater soluble chromogens should not be used with aqueous media Nonaqueous mounting media is not compatible with water; therefore, the samples must be first dehydrated with a series of ethanol and xylene washes:

39. Controls for interpretation and accuracy Positive Negative