1. Histology 1.2.: Immunohistochemistry

2. Immunohistochemistry uses the principle of immunity: During development the immune system recognizes foreignDuring development the immune system recognizes foreign proteins as antigens If foreign proteins invade the body, this evokes immune responseIf foreign proteins invade the body, this evokes immune response One type of immune response is the production of highly specificOne 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 localizationImmunocytochemistry 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.

3. 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

4. An example: pre-embedding immunohistochemical reaction: 1.Antigen (green triangle)-antibody binding in the tissue 2. Antigen-antibody binding between the primary antibody between the primary antibody and the secondary antibody and the secondary antibody labelled with either a gold labelled with either a gold particle, or a fluorescent dye, particle, or a fluorescent dye, or an enzyme catalysing or an enzyme catalysing a chromogen reaction a chromogen reaction

5. The results: Epithelial cells infected with influensa viruses (brown dots) in the wall of a bronchus in the lung

6. B Nerve cells containing the enzyme nitrogen monoxide synthase (DAB reaction, brown precipitate) Endothelial cell culture: Red fluorescence: actin cytoskeleton Green fluorescence: tubulin Blue: DAPI staining of the nucleus (not immune staining) (not immune staining)

7. IMMUNFLUORESCENCE GAD-GFP and NPY in fluo microscope GAD-GFP and enk, confocal micr.

8. 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. 100.000 times smaller than that of light. - the electron beam can be manipulated with electromagnetic field just like the light with glass lenses field just like the light with glass lenses 1931:Ernst Ruska built the first electron microscope

9. The transmission electron microscope (TEM)

11. Electron source: triode gun 1. filament: tungsten, heated up to 2700 o C: 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 get through it with a speed of several 100.000 km/secMagnification: 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)

12. 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 o C) temperature, polymerises at 56 o C) 6. Preparation of semithin (0.5  m) and ultrathin (60-90 nm) sections Staining/contrasting with lead citrate. Staining/contrasting with lead citrate.

13. The ultramicrotom:

14. The electron micrograph nucleus

15. 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) has to be covered with a thin layer of metal (e.g. gold) - Secondary electrons are detected and turned into an electrical signal. signal. - In the monitor electrical signal is turned into light to produce an image. an image.

16. SEM images : Red and white blood cells Blood clotting

17. 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  m vibratome sections

18. Further steps after the immunoreaction: -contrasting: buffered 1 % OsO430-60 min -Dehydration in ascending series of ethanol10-10 min (70 % ethanol is saturated with uranyl acetate) - Intermedier solvent: propylene oxide10 min - Durcupan : propylene oxide 1:1 30 min - Durcupan resinovernight - Mounting on glass slide in Durcupan resin - Polimerization 56 o C-onone day - re-embedding for ultrathin sectioning - Preparation of ultrathin sections (60-90 nm) in ultramicrotome - Contrasting with lead citrate2-10 min - View in electron microscope

19. Light microscopic levelElectron microscopic level

20. Postembedding immunogold labelling: - Carried out on ultrathin sections - Secondary antibody is decorated with a colloidal gold particle Localization of gonadotrop hormonpresynaptic membrane protein