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Methods in histology

Methods in histology. Microscopy. Organization of the practices How microscopic slides are made Microscop e Staining Observation of slides. Observation of the live cells. Unicellular organisms Metazoas : germ cell s, blood cells, cells in tissue cultures

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Methods in histology

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  1. Methods in histology Microscopy

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

  3. 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

  4. 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 (AUTOLYZE) • Tissue block for fixation must not exceed (be bigger than)1cm3 ( for light microscopy)

  5. Fixation • Fixation stops the metabolic events in the cell either by their reduction or by denaturation (destruction) of enzymes. • Physical methods: • Heat (microwave oven) • Freezing (in liquid nitrogen; -170oC) • Chemical methods: • Immersion (into fixative) • Perfusion (into vessels)

  6. Chemical fixation

  7. Fixatives

  8. Embedding and cutting • Tissue have to be harden or stabilize 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“

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

  10. Cutting • Tissue is cutin slides of one cell layer, it means 4-10mm. 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

  11. Mikrotomes

  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. Staining • For staining we use jars or automatic machines.

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

  15. Procedure

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

  17. 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 mm. • Magnification – 1000-1500 times

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

  19. 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

  20. Haematoxylin - eosin

  21. 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

  22. AZAN

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

  24. Weigert - van Gieson Nalepení řezů na podložní sklo

  25. Weigert - van Gieson

  26. Haematoxylin - eosin

  27. Weigert-van Gieson

  28. Green Masson trichrome

  29. AZAN

  30. 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

  31. Yellow Masson trichrome

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

  33. Weigert resorcin - fuchsin

  34. Heidenhain iron haematoxylin • Heidenhain iron hematoxylin stains nucleus as well as cytoplasma gray-black. • It is used for staining of muscles; and in parasitology for detection of worms in tissue.

  35. Heidenhain iron haematoxylin

  36. Heidenhain iron haematoxylin

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

  38. Silver method

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

  40. Cresyl violet

  41. Cresyl violet

  42. Results of staining

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

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