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Special methods in histology 195 SFST SEM-řádkovací elektronový mikroskop Umožňuje zobrazení povrchu studovaných objektů Má menší rozůlišovací schopnost než TEM Sampling Sampling of tissue and cells : From the live organism (BIOPSY) From the corpse (NECROPSY) Fixation must be made otherwise enzymes and germs (bacterias) destroy the cells (AUTOLYSIS) Tissue block for fixation must not exceed (be bigger than) 1cm3 ( for light microscopy) Or 1mm3 ( for electro microscopy) Fixation Fixation stops the metabolic events in the cell either by denaturation (destruction) of enzymes or reduction of their activity Physical methods: Heat (microwave oven) Freezing (in liquid nitrogen; -170oC) Chemical methods: Immersion (into fixative) Perfusion (into vessels) Chemical fixation Aldehydes Formaldehyde, glutaraldehyde Alcohols Methanol, ethanol Acids Acetic acid, trichloracetic acid, picric acid Salts of heavy metals Mercury, osmium, chromium Fixatives Formaldehyde 4% Bouin fluid trinitrophenol, formaldehyde, acetic acid Susa mercuric chloride, natrium chloride,acetic acid, trichloracetic acid, formaldedyde Zenker fluid Carnoy Mercuric chloride, potassium bichromate, natrium sulphate, acetic acid ethanol, chloroform, acetic acid Methacarn methanol, chloroform, acetic acid Embedding and cutting Tissue have to be harden or stabilized for cutting by embedding in special medias (paraffin, celloidin). These medias are not mixable with water therefore we remove water from the tissue by alcohols (dehydratation) and then we replete it by solvent of embedding medium (xylene, toluene, acetone), which procedure is named „clearing“ Cutting Tissue is cut in slides of one cell layer, it means m. Tissue is translucent and „well-readable“ in this case Devices that are used for cutting are called microtomes. Tissue slices are put on slide. They are stretched out by heat, and stick by egg whiteglycerin Microtomes Staining Staining facilitates to distinguish tissue and cell components The majority of dyes are water-soluble, therefore we have to remove paraffin (dewax). Slide is covered by cover slip after the staining. Resins (Canadian or synthetic resins) are used as glue. The slide is long lasting. Permanent slide Water is removed from tissue after staining Cover slip is stick by resin Permanent slide is made Resolution Resolving power is the smallest distance between two particles at which we are able to distinguish them as two separate objects Resolving power for light microscopy is 0,2 m. Magnification – 1000-1500 times Resolving power for electron microscopy is 0,2 nm Staining General staining Haematoxylin - eosin Masson trichrome Weigert - van Gieson Heidenhain iron haematoxylin Selective Weigert resorcin fuchsin Silver methods Haematoxylin - eosin Haematoxylin stains acidic components of cell (basophilic structures) – DNA, RNA, ie. Nucleus, nucleolus, ribosomes a rough endoplasmatic reticulum Eosin stains basic structures of cell (acidophilic, eosinophilic) – that are predominantly proteins, ie. cytoplasma, mitochondrias, smooth endoplasmatic reticulum, and collagen in extracellular matrix Haematoxylin - eosin Results of staining Staining Dyes Nucleus Collagen Elastic Muscle Notice Haematoxylin-eosin Haematoxylin Eosin Blue to blac pink pink Weigert – van Gieson Weigert haematoxylin Saturn red Trinitrofenol Brown red yellow Acid fuchsin can be used instead of Saturn red, all tissues are yellow except of collagen AZAN Azocarmine aniline blue Orange G red blue Orange – red Red - erythrocytes blue- mucus Blue Masson trichrome Haematoxylin Acid fuchsin Anilin blue blue to black blue red Red- erythrocytes Blue - mucus Yellow Masson trichrome Haematoxylin Erythrosin saffron blue to black yellow red Red – erythocytes Green Masson trichrome Haematoxylin Acid fuchsin Light green blue to black green red red - erythrocytes Weigert resorcinfuchsin Resorcin Fuchsin Silver AgNO3 grey-black Reticular fibres- black Heidenhain iron haematoxylin HIH Heidenhain iron haematoxylin violet brown brown to black grey- black AZAN Azocarmine stains nuclei (red) Aniline blue stains collagen fibres and mucus (blue) Orange G stains cytoplasma, muscles (orange) Weigert van Gieson Weigert haematoxylin nucleus is brown Saturn red stains collagen fibres and mucus (red) Trinitrophenol (picric acid) stains cytoplasma and muscles (yellow) Green Masson Trichrome Hematoxylin stains nuclei blue Light green stains collagen green Acid fuchsin stains muscle tissue red Weigert resorcin - fuchsin Resorcin –fuchsin stains only elastic fibres Elective staining for elastic fibres Heidenhain iron haematoxylin Heidenhain iron haematoxylin stains nucleus as well as cytoplasma gray-black. It is used for staining of muscles; and in parasitology for detection of worms in tissue. Silver methods Silver stains reticular and collagen fibres in brown to black. Silver methods are used for staining of neurons in neurohistology. Electron microscopy TEM Method of ultra-thin section Sampling Fixation (glutaraldehyde, paraformaldehyde and osmium oxide) Embedding (epoxide, polyester and acrylate resins) Polymeration Cutting - thickness 50-60nm Contrasting (osmium, uran, tungsten) Observation TEM Method of negative staining Corpuscle is surrounded by electron-dense substance – phospho-tungsten acid or uranyl acetate = dense background, particles are light Used for detection of viruses Scanning electron microscopy SEM It allows to demonstrate the surface of cells It has lower reolving power than TEM Histochemistry It uses chemical and histochemical reaction for the detection of elements or compounds in situ in cells and tissues Histochemistry Catalytic histochemistry Affinity histochemistry Detection of elements or compounds Elements: Hg, Pb, Fe, Ca, Zn and their salts Perls reaction –detection of Fe2+ Fe2+ (HCl) and potassium ferrocyanide. Product of reaction is Prussian blue Detection of organic compounds Carbohydrates: polysaccharides (glycogen) glycoproteins and proteoglycans, glycolipids (PAS reaction – HIO4 + Schiff reagent) Lipids (lipid soluble dyes) Sudan dyes: Sudan black, Sudan IV, oil red Catalytic histochemistry It allow detection of enzymes (enzymatic activities) in tissues and cells Used for: Research: localization of enzymes in cell Diagnostic: celiac disease They serves as markers for visualization in affinity histochemistry Catalytic histochemistry 1. histochemical reaction Tissue with Enzyme + Substrate = Product 2. reaction – visualisation Coloured and insoluble compound arises from colourless product of first reaction Affinity histochemistry Immunohistochemistry – detection of proteins (glycoproteins) by the binding of the specific antibody to the antigen Lectin histochemistry –detection of mono-, di-, tri-, i polysaccharides in the complex molecules by binding of lectins to the saccharides In situ hybridization – detection of specific sequence of nucleoids in DNA or m-RNA by the binding of complementary chain of probe Monoclonal and polyclonal antibodies Antibody binds to specific place on protein – epitop Antibodies– polyclonal monoclonal Immunohistochemistry is used for : Diagnostic of tumors and other illnesses in pathology The most important antigens: Intermediate filaments, CD antigens, hormones, estrogen and progesteron receptor, melanoma antigens, S-100 protein, PSA (prostatic specific antigen),proliferation specific antigens: PCNA, p53 protein, KI-67 Research