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Special methods in histology
195 SFST
SEM-řádkovací elektronový mikroskopUmožňuje 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
Mercury, osmium, chromium
Salts of heavy metals
Acetic acid, trichloracetic acid, picric acid
Acids
Methanol, ethanolAlcohols
Formaldehyde, glutaraldehyde
Aldehydes
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%
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 white-glycerin
Microtomes
StainingStaining 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
grey- blackbrown to black
Heidenhain ironhaematoxylin
Heidenhain iron haematoxylin HIH
Reticular fibres- blackgrey-blackbrownAgNO3Silver
violetResorcinFuchsin
Weigert resorcin-fuchsin
red - erythrocytesredgreenblue to black
HaematoxylinAcid fuchsinLight green
Green Masson trichrome
Red – erythocytesred yellowblue to black
HaematoxylinErythrosinsaffron
Yellow Masson trichrome
Red- erythrocytesBlue - mucus
redblue blue to black
HaematoxylinAcid fuchsinAnilin blue
Blue Masson trichrome
Red - erythrocytesblue- 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 redTrinitrofenol
Weigert – van Gieson
pinkpinkBlue to blac
HaematoxylinEosin
Haematoxylin-eosin
NoticeMuscleElasticCollagenNucleusDyesStaining
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
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
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
HistochemistryCatalytic histochemistryAffinity 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 compoundsCarbohydrates:
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 cellDiagnostic: celiac diseaseThey 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