1. Special methods in histology 195 SFST

2. SEM-řádkovací elektronový mikroskop Umožňuje zobrazení povrchu studovaných objektů Má menší rozůlišovací schopnost než TEM

3. 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) 1cm 3 ( for light microscopy)‏ Or 1mm 3 ( for electro microscopy)

4. 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; -170 o C)‏ Chemical methods:  Immersion (into fixative)‏  Perfusion (into vessels)

5. Chemical fixation Mercury, osmium, chromium Salts of heavy metals Acetic acid, trichloracetic acid, picric acid Acids Methanol, ethanolAlcohols Formaldehyde, glutaraldehyde Aldehydes

6. Fixatives methanol, chloroform, acetic acidMethacarn ethanol, chloroform, acetic acidCarnoy Mercuric chloride, potassium bichromate, natrium sulphate, acetic acid Zenker fluid mercuric chloride, natrium chloride,acetic acid, trichloracetic acid, formaldedyde Susa trinitrophenol, formaldehyde, acetic acid Bouin fluid Formaldehyde 4%

7. 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“

8. 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 white- glycerin

9. Microtomes

10. 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.

11. Permanent slide Water is removed from tissue after staining Cover slip is stick by resin Permanent slide is made

12. 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

13. Staining General staining Haematoxylin - eosin Masson trichrome Weigert - van Gieson Heidenhain iron haematoxylin Selective Weigert resorcin fuchsin Silver methods

14. 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

15. Haematoxylin - eosin

16. Results of staining grey- blackbrown to black Heidenhain iron haematoxylin Heidenhain iron haematoxylin HIH Reticular fibres- blackgrey-blackbrownAgNO 3 Silver violetResorcin Fuchsin Weigert resorcin- fuchsin red - erythrocytesredgreenblue to black Haematoxylin Acid fuchsin Light green Green Masson trichrome Red – erythocytesredyellowblue to black Haematoxylin Erythrosin saffron Yellow Masson trichrome Red- erythrocytes Blue - mucus red blue blue to black Haematoxylin Acid fuchsin Anilin blue Blue Masson trichrome Red - erythrocytes blue- mucus Orange – redblueredAzocarmine aniline blue Orange G AZAN Acid fuchsin can be used instead of Saturn red, all tissues are yellow except of collagen yellowredBrownWeigert haematoxylin Saturn red Trinitrofenol Weigert – van Gieson pink Blue to blac Haematoxylin Eosin Haematoxylin-eosin NoticeMuscleElasticCollagenNucleusDyesStaining

17. AZAN Azocarmine stains nuclei (red)‏ Aniline blue stains collagen fibres and mucus (blue)‏ Orange G stains cytoplasma, muscles (orange) Red blood cells are red - erythrocytes

18. Weigert van Gieson Weigert haematoxylin nucleus is brown Saturn red stains collagen fibres and mucus (red)‏ Trinitrophenol (picric acid) stains cytoplasma and muscles (yellow)‏ Acid fuchsin can be used instead of Saturn red, all tissues are yellow except of collagen

19. Green Masson Trichrome Hematoxylin stains nuclei blue Light green stains collagen green Acid fuchsin stains muscle tissue red

20. Weigert resorcin - fuchsin Resorcin –fuchsin stains only elastic fibres Elective staining for elastic fibres

21. 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.

22. Silver methods Silver stains reticular and collagen fibres in brown to black. Silver methods are used for staining of neurons in neurohistology.

23. Electron microscopy TEM

24. 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

25. TEM

26. 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

27. Scanning electron microscopy SEM It allows to demonstrate the surface of cells It has lower reolving power than TEM

28. 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

29. Detection of elements or compounds Elements: Hg, Pb, Fe, Ca, Zn and their salts Perls reaction –detection of Fe 2+ Fe 2+ (HCl) and potassium ferrocyanide. Product of reaction is Prussian blue ‏

30. Detection of organic compounds Carbohydrates: polysaccharides (glycogen) glycoproteins and proteoglycans, glycolipids (PAS reaction – HIO 4 + Schiff reagent) Lipids (lipid soluble dyes)‏ Sudan dyes: Sudan black, Sudan IV, oil red

31. 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

32. Catalytic histochemistry 1. histochemical reaction Tissue with Enzyme + Substrate = Product 2. reaction – visualisation Coloured and insoluble compound arises from colourless product of first reaction

33. 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

34. Monoclonal and polyclonal antibodies Antibody binds to specific place on protein – epitop Antibodies– polyclonal monoclonal

37. 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