Transcript Haematoxylin

Special methods in
histology
195 SFST
SEM-řádkovací elektronový
mikroskop
Umožňuje zobrazení
povrchu studovaných
objektů
Má menší rozůlišovací
schopnost než TEM
Sampling
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Sampling of tissue and cells :
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From the live organism (BIOPSY)
From the corpse (NECROPSY)
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Fixation must be made otherwise enzymes and
germs (bacterias) destroy the cells (AUTOLYSIS)
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Tissue block for fixation must not exceed (be
bigger than) 1cm3 ( for light microscopy)
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Or 1mm3 ( for electro microscopy)
Fixation
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Fixation stops the metabolic events in the cell
either by denaturation (destruction) of
enzymes or reduction of their activity
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Physical methods:
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Heat (microwave oven)
Freezing (in liquid nitrogen; -170oC)
Chemical methods:
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Immersion (into fixative)
Perfusion (into vessels)
Chemical fixation
Aldehydes
Formaldehyde,
glutaraldehyde
Alcohols
Methanol, ethanol
Acids
Acetic acid, trichloracetic
acid, picric 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
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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
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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 whiteglycerin
Microtomes
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.
Permanent slide
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Water is removed from tissue after staining
Cover slip is stick by resin
Permanent slide is made
Resolution
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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
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General staining
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Haematoxylin - eosin
Masson trichrome
Weigert - van Gieson
Heidenhain iron haematoxylin
Selective
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Weigert resorcin fuchsin
Silver methods
Haematoxylin - eosin
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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
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
AZAN
Azocarmine stains nuclei
(red)
Aniline blue stains
collagen fibres and
mucus (blue)
Orange G stains
cytoplasma, muscles
(orange)
Weigert van Gieson
Weigert haematoxylin
nucleus is brown
Saturn red stains
collagen fibres and
mucus (red)
Trinitrophenol (picric
acid) stains cytoplasma
and muscles (yellow)
Green Masson Trichrome
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Hematoxylin stains
nuclei blue
Light green stains
collagen green
Acid fuchsin stains
muscle tissue red
Weigert resorcin - fuchsin
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Resorcin –fuchsin
stains only elastic
fibres
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Elective staining
for elastic fibres
Heidenhain iron haematoxylin
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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
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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
Histochemistry
Catalytic histochemistry
Affinity 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
compounds
Carbohydrates:
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 cell
Diagnostic: celiac disease
They 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