1. Methods in histology Microscopy Maňáková 2009

2. Content of practices Organization of the practices How microscopic slides are made Microscope Staining Observation of slides

3. What you need? The basic knowledge is needed Pen and paper Textbook needed for histology: Junqueira, Carneiro: Basic Histology Moore,Persaud: Before we are born or Human Embryology

4. Observation of the live cells Unicellular organisms Metazoas: germ cells, blood cells, cells in tissue cultures Observation is possible by special microscope (phase contrast microscopy) or using supravital staining

5. 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)‏

6. 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)

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

8. 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%

9. 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“

10. Embedding Tissue can be proceed in beakers in thermostat (in small laboratory)‏ Automatic embedding machines serve for the pathologic department running

11. 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

12. 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.

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

14. Procedure Resins or glycerin -gelatineResins Sometime dehydratation and clearing Dehydratation and clearing Histochemical reactions predominantly Staining, histochemical reaction Washing Sometime short fixationDewaxing and rehydratation Sticking on slide Cutting in cryostatCutting Embedding in paraffin Clearing by solvents Dehydratation by alcohols Washing Freezing at – 170 o CFixation Cryostat slidesParaffin slides

15. Microscope Stative Adjustment knob Optic system: Oculars Objectives Condensor Light 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

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

17. 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

18. Haematoxylin - eosin

19. 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

20. AZAN

21. 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

22. Weigert - van Gieson Nalepení řezů na podložní sklo

23. Weigert - van Gieson

24. Haematoxylin - eosin

25. Weigert-van Gieson

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

27. Green Masson trichrome

28. AZAN

29. Yellow Masson trichrome Haematoxylin stains nucleus blue to black Erythrosin stains cytoplasma and muscles red Saffron stains collagen fibres yellow Red blood cells are red

30. Yellow Masson trichrome

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

32. Weigert resorcin - fuchsin

33. 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.

34. Heidenhain iron haematoxylin

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

36. Silver method

37. Cresyl violet Cresyl violet stains DNA and RNA, ei. nucleus, nucleolus and rough endoplasmatic reticulum Staining is used in neurohistology

38. Cresyl violet

40. 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

41. What is necessary to know What is fixation? Why it is performed? How we make slide? Overview. Basic methods for staining. Why we stain tissues by various staining methods? Haematoxylin –eosin staining Light microscopy resolution (0,2  m)‏