Transcript Staining
Methods in histology Microscopy Organization of the practices How microscopic slides are made Microscope Staining Observation of slides 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 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) 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) Chemical fixation Aldehydes Formaldehyde, glutaraldehyde Alcohols Methanol, ethanol Acids Acetic acid, trichloracetic acid, pikric 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 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“ Embedding Tissue can be proceed in beakers in thermostat (in small laboratory) Automatic embedding machines serve for the pathologic department running Cutting Tissue is cut in 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 whiteglycerin Mikrotomes 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. Staining For staining we use jars or automatic machines. Permanent slide Water is removed from tissue after staining Cover slip is stick by resin Permanent slide is made Procedure Paraffin slides Cryostat slides Fixation Freezing at – 170 oC Washing Dehydratation by alcohols Clearing by solvents Embedding in paraffin Cutting Cutting in cryostat Sticking on slide Sticking on slide Dewaxing and rehydratation Sometime short fixation Washing Staining, histochemical reaction Histochemical reactions predominantly Dehydratation and clearing Sometime dehydratation and clearing Resins Resins or glycerin -gelatine Microscope Stative Adjustment knob Optic system: Oculars Objectives Condensor Light 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 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 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 AZAN Weigert van Gieson Weigert haematoxylin nucleus is brown Saturn red stains collagen 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 Weigert - van Gieson Nalepení řezů na podložní sklo Weigert - van Gieson Haematoxylin - eosin Weigert-van Gieson Green Masson trichrome AZAN 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 Yellow Masson trichrome Weigert resorcin - fuchsin Resorcin –fuchsin stains only elastic fibres Elective staining for elastic fibres Weigert resorcin - fuchsin 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. Heidenhain iron haematoxylin Heidenhain iron haematoxylin Silver methods Silver stains reticular and collagen fibres in brown to black. Silver methods are used for staining of neurons in neurohistology. Silver method Cresyl violet Cresyl violet stains DNA and RNA, ei. nucleus, nucleolus and rough endoplasmatic reticulum Staining is used in neurohistology Cresyl violet Cresyl violet 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 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)