Special methods in histologyvyuka-data.lf3.cuni.cz/CVSE1M0001/639 special... · Fixatives Methacarn...
Transcript of Special methods in histologyvyuka-data.lf3.cuni.cz/CVSE1M0001/639 special... · Fixatives Methacarn...
Special methods in
histology
639
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 electron 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
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
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
endoplasmic reticulum
Eosin stains basic structures of cell
(acidophilic, eosinophilic) – that are
predominantly proteins, ie. cytoplasma,
mitochondrias, smooth endoplasmic
reticulum, and collagen in extracellular matrix
Haematoxylin - eosin
AZAN
Azocarmine stains nuclei
(red)
Aniline blue stains
collagen fibres and
mucus (blue)
Orange G stains
cytoplasm, muscles
(orange)
Red blood cells are
Weigert van Gieson
Weigert haematoxylin
nucleus is brown
Saturn red stains
collagen fibres and
mucus (red)
Trinitrophenol (picric
acid) stains cytoplasm
and muscles (yellow)
Acid fuchsin can be
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 cytoplasm
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.
Results of staining
grey- blackbrown to
black
Heidenhain iron
haematoxylin
Heidenhain iron
haematoxylin HIH
Reticular fibres- blackgrey-blackbrownAgNO3Silver
violetResorcin
Fuchsin
Weigert resorcin-
fuchsin
red - erythrocytesredgreenblue to
black
Haematoxylin
Acid fuchsin
Light green
Green Masson
trichrome
Red – erythocytesred yellowblue to
black
Haematoxylin
Erythrosin
saffron
Yellow Masson
trichrome
Red- erythrocytes
Blue - mucus
redblueblue to
black
Haematoxylin
Acid fuchsin
Anilin blue
Blue Masson
trichrome
Red - erythrocytes
blue- 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 red
Trinitrofenol
Weigert – van
Gieson
pinkpinkBlue to
blac
Haematoxylin
Eosin
Haematoxylin-eosin
NoticeMuscleElasticCollagenNucleusDyesStaining
Permanent slide
Water is removed from tissue after staining
Cover slip is stick by resin
Permanent slide is made
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 Fe3+
Fe3+ (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
Conditions:
To maintain the enzymatic activity and structure of tissue and cells, we use cryostat sections
Fixation degreases or inhibits completely the enzymatic activity
pH, temperature, substrate in abundance
Activators and inhibitors
For 200 enzymes are available special procedures - protocols
Methods for visualization in
catalytic histochemistry
Precipitation
Metal salt capture methods (Cobalt, Lead, cerium)
Diazonium salt methods
Indigogenic methods
Tetrazolium salt methods
DAB- diaminobenzidine methods
Affinity histochemistry
Immunohistochemistry – detection of proteins (glycoproteins) by the bond of the specific antibody to the antigen
Lectin histochemistry –detection of mono-, di-, tri-, or even polysaccharides in the complex molecules by bond of lectins to the saccharides
In situ hybridization – detection of specific sequence of nucleoids in DNA or m-RNA by the bond of complementary chain of probe
Immunohistochemistry
Antibody binds to specific place on protein –
epitop
Antibodies– polyclonal
monoclonal
Markers
Antibodies (lectins and probes, too) have
to be visualized by markers: Fluorochromes (FITC, rhodamine)
Biotin
Enzymes (HRP, alP) – catalytic histochemistry is
used for their visualization
Colloidal gold particles
Isotopic probes
Ferritin, digoxigenin
Immunohistochemistry
Direct reaction Ag + AB
Indirect reaction Ag + AB1 + AB2
PAP reaction
Peroxidase –
Anti-peroxidase
Signal amplification
More molecules of
enzyme mean much
product and heavier
signal
ABC reaction
Biotin is marker of
secondary antibody
It reacts with avidin
that is bound to the
enzyme
Signal is amplified
Insulin
Cytokeratins
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
Lectins
Lectins are proteins or glycoproteins that agglutinate cells and/or precipitate complex carbohydrates. The binding is highly specific. Lectins are isolated from a wide variety of natural sources including plants, fungi, bacteria and vertebrates.
Application: blood grouping
mitogenic stimulation of cells (lymphocytes)
histochemical studies
Electron microskopy -TEM
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
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
Semithin section X Ultrathin section
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
Cryofracture – freeze fracture
Frozen tissues are fractured, coated by metal dust, observed in TEM
Structure of membranes
Scanning electron microscopy SEM
It allows to demonstrate the surface of cells
It has lower reolving power than TEM