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Histology 1.2.: Immunohistochemistry Immunohistochemistry uses the principle of immunity: • During development the immune system recognizes foreign proteins as antigens • If foreign proteins invade the body, this evokes immune response • One type of immune response is the production of highly specific molecules against the foreign proteins. These are called antibodies, binding with high affinity to the antigens • Immunocytochemistry utilizes these antibodies for the localization of tissue components Production of antibodies: 1. A tissue constituent is extracted from the body of X animal (e.g. goat), and purified 2. This material is injected into the bloodstream of Y animal (e.g. rabbit), where it behaves as antigen and evokes immune response, thus, production of highly specific antibodies 3. The antibody can be extracted from the blood of Y animal, purified and characterized. Preparation of tissues for immunohistochemistry: 1. Collection of samples (tissue blocks from experimental animals, biopsy, smears, etc.) Fixation: - immersion (drop the tissue block into fixative) - perfusion through the heart Perfusion: 1. Deep anaesthesia (Nembutal, etc.) 2. Cannule introduced to the left ventricle or into the aorta 3. Wash out the blood with a saline 4. Fix with paraformaldehyde and/or glutaraldehyde 5. Removal of the wanted tissue or organ immersion-fixed for some hours 6. Sectioning 7. Incubation of sections An example: pre-embedding immunohistochemical reaction: 1. Antigen (green triangle)-antibody binding in the tissue 2. Antigen-antibody binding between the primary antibody and the secondary antibody labelled with either a gold particle, or a fluorescent dye, or an enzyme catalysing a chromogen reaction The results: Epithelial cells infected with influensa viruses (brown dots) in the wall of a bronchus in the lung Nerve cells containing the enzyme nitrogen monoxide synthase (DAB reaction, brown precipitate) B Endothelial cell culture: Red fluorescence: actin cytoskeleton Green fluorescence: tubulin Blue: DAPI staining of the nucleus (not immune staining) IMMUNFLUORESCENCE GAD-GFP and NPY in fluo microscope GAD-GFP and enk, confocal micr. The electron microscope Brief history: 1920: physicists discovered that accelerated electrons behave in vacuum jut like light - they travel in straight lines and their wavelength is about 100.000 times smaller than that of light. - the electron beam can be manipulated with electromagnetic field just like the light with glass lenses 1931: Ernst Ruska built the first electron microscope The transmission electron microscope (TEM) Electron source: triode gun 1. filament: tungsten, heated up to 2700oC: emits electron cloud 2. Wehnelt cylinder: bunches the electrons into finely focused point 3. anode: has a hole in it so that the accelerated electron beam get through it with a speed of several 100.000 km/sec Magnification: with the help of electromagnetic lenses: changing the strength of the current within the coils changes the magnification Image formation: the focussed electron beam reaches the extremely thin specimen (60-90 nm), passes through it and the image is projected to a fluorescent screen the specimen has to be treated with heavy metal salts in order to get contrasty image („staining”=contrasting) Preparation fo tissues for electron microscopy: 1. Fixation: buffered solutions of paraformaldehyde and glutaraldehyde (immersion and perfusion) 2. Staining/contrasting with osmium tetroxide 3. Dehydration: in ascending series of ethanol (50%-100%) Staining/contrasting with 70 % ethanol saturated with uranyl acetate 4. Intermediate solvent: propylene oxide 5. Embedding: in synthetic resins e.g. Durcupan ACM (liquid at room temperature, polymerises at 56 oC) 6. Preparation of semithin (0.5 mm) and ultrathin (60-90 nm) sections Staining/contrasting with lead citrate. The ultramicrotom: The electron micrograph nucleus Scanning electron microscope (SEM) Suitable to observe the surface of tissue components Parts of SEM: Electron optical column (short with 3 lenses) Specimen chamber Works like the tv screen: - The electron beam hits the surface of the specimen which has to be covered with a thin layer of metal (e.g. gold) - Secondary electrons are detected and turned into an electrical signal. - In the monitor electrical signal is turned into light to produce an image. SEM images : Red and white blood cells Blood clotting Pre-embedding immunocytochemistry at electron microscopic level: Its steps are similar to those of light microscopic ICC but: - Triton X-100 detergent is not allowed to use - Instead Triton X-100 freeze-thaw in liquid nitrogen helps the penetration of antibodies - The immunoreaction is carried out on 60-80 mm vibratome sections Further steps after the immunoreaction: -contrasting: buffered 1 % OsO4 30-60 min -Dehydration in ascending series of ethanol 10-10 min (70 % ethanol is saturated with uranyl acetate) - Intermedier solvent: propylene oxide 10 min - Durcupan : propylene oxide 1:1 30 min - Durcupan resin overnight - Mounting on glass slide in Durcupan resin - Polimerization 56 oC-on one day - re-embedding for ultrathin sectioning - Preparation of ultrathin sections (60-90 nm) in ultramicrotome - Contrasting with lead citrate 2-10 min - View in electron microscope Light microscopic level Electron microscopic level Postembedding immunogold labelling: - Carried out on ultrathin sections - Secondary antibody is decorated with a colloidal gold particle Localization of gonadotrop hormon presynaptic membrane protein