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Methods in histologyMicroscopyMaňáková
2009
Content of practices
Organization of the practices
How microscopic slides are made Microscope Staining Observation of slides
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
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 (AUTOLYSIS)
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 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“
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 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
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
Procedure
Resins or glycerin -gelatineResins
Sometime dehydratation and clearingDehydratation and clearing
Histochemical reactions predominantly
Staining, histochemical reaction
Washing
Sometime short fixationDewaxing and rehydratation
Sticking on slideSticking on slide
Cutting in cryostatCutting
Embedding in paraffin
Clearing by solvents
Dehydratation by alcohols
Washing
Freezing at – 170 oCFixation
Cryostat slidesParaffin slides
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
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)
Trinitrophenol (picric acid) 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
Hematoxylin stains nuclei blue Light green stains collagen green Acid fuchsin stains muscle tissue red
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 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.
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
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
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)