Download - Hi Techniques 2010
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HISTOLOGICAL TECHNIQUES
A) LIGHT MICROSCOPY
B) FLUORESCENT MICROSCOPY
C) ELECTRON MICROSCOPY
D) AUTORADIOGRAPHY
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- observation of unstained structures in dark field or using phase contrast (tissue
cultures)
- can use frozen, paraffin-embedded, vibratome, ground sections, etc.
- examination of cells and tissues after staining with histological dyes
- impregnation
- histochemical methods
A) LIGHT MICROSCOPY:
Histochemistry = the science devoted to chemical detection of components in
tissues. Plant or animal tissues must be fixed with fixatives that do not cause
changes in the localization of detected components and do not decrease their
reactivity. A chemical reaction is performed on a section; then its intensity and
location is examined with the use of a microscope.
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- Enzymatic histochemistry: detection of enzymatic activity usingtheir reaction with substrates; the resulting (coloured) reaction product
is insoluble and accumulates in the site of enzymatic activity
(peroxidase, alkaline phosphatase, cholinesterase, peptidase, etc.).
Cytochemistry = the science that examines location of chemical
components and reactions in cells (obtained for example: from tissuecultures, sediments or centrifugates of body fluids, from blood or
bone marrow smears or tissue suspensions). To detect components in
cytochemistry, the same procedures are used as in histochemistry.
- Classical histochemistry: eg. detection of Fe3+ in Perls reaction:
4 Fe3++ 3 K4[Fe(CN)6] 12 K+ + Fe4[(Fe(CN)6]3 (Berlin blue);
detection of glycogene (PAS reaction, Bests carmine), lipid
detection (Sudan III, IV), detection of mucopolysaccharides (colloidiron according to Hale-Mller), detection of DNA (Feulgens nuclear
reaction).
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- Lectin histochemistry:
Lectins = plant-derived proteins with high and specific affinity
(Ka=103-104l/mol) for glycoproteins. For example, lectins labelled
with enzymes or fluorochromes can be used for identification of
certain sacccharide components in membrane glycoproteins which
are typical for a given cellular population (sacccharide components
of membrane glycoproteins undergo changes in the process of
cellular differentiation, during transformation of neoplastic cells,
etc.).
Example: Lectin: Griffonia simplicifolia - microglia
Ulex europeaus - identifies capillaries
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Immunohistochemistry: a method that enables detection of antigens
in tissues with the use of labelled specific antibodies. The reaction ishighly specific and sensitive. Antibodies bind antigens with high
specifity and affinity (Ka= 105-1011l/mol) and form the antigen-
antibody complex. Antibodies are generated by immunization of
animals with antigens, e.g. proteins isolated from human cells -
detection of proliferation markers, intermediate filaments, membrane
molecules, enzymes, etc.
Antigen = macromolecular biological compound (protein, poly-
saccharide, nucleic acid, etc.), that is recognized by immune system
as foreign and evokes development of immune reaction (i.e.generation of specific antibodies). Antigen carries the so-called
antigen determinants (epitopes) = characteristic groups of atoms on
the surface of an antigen that is the target of the immune response (it
is bound by antibody).
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tissue/cellular antigen
antibody with a marker
DIRECT METHOD
tissue antigen
primary antibody
(unlabelled)
secondary antibody(anti-immunoglobulin)labelled with a marker
INDIRECT TWO-STEP
METHOD
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tissuetissue//cellularcellular antigenantigen
primary antibody
AVIDIN-BIOTIN
METHOD LAB
secondary antibody
labelled with biotin
avidin labelled with
a marker
Affinity (Ka-1015
l/mol)
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Example.: DNA: G-C A-T
probe: cag gat tcc ctc gga tct -
3'- cac tca gtc cta agg gag cct aga gat cgt - 5'
In situ hybridization: method that enables specific detection of a
nucleotide sequence in the DNA or mRNA. ISH utilizes a
labelled probe (eg. oligo-nucleotides) that binds in a specific
manner to complementary nucleotide chain in the nucleic acid. It
is suitable for detection of proviruses in cells or for detection of
gene transcription.
chain of the DNA:
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B) FLUORESCENT MICROSCOPY- examination of tissues using a special (fluorescent)
microscope. In this microscope, a sample is exposed to alight of a short wavelength (ie. high energy: UV, blue,
green); if a tissue section contains a fluorochrome, its
molecule is excited under UV light and emits visible light of
a characteristic (longer) wavelength (ie. typical colour -
visible light) - picture of structures that emit this light can beobserved in a microscope.
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reflected lightis absorbed
emitted light
(visible)
barrier filter
FLUORESCENTMICROSCOPE
(epifluorescence)
histological slide
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- certain fluorochromes (fluorescein = green, rhodamine = red) can
be used to label other molecules eg. antibodies
(immunofluorescence), avidin, protein A, lectins, oligonucleotide
probes (fluorescent in situ hybridization = FISH).
confocal microscope
- autofluorescence = primary (native) fluorescence (chlorofyl,
tetracyclin, lipofuscin etc.)
- to visualize structures that do not exhibit primary fluorescence,
fluorescent dyes (fluorochromes) must be utilized; light emitted by
these dyes is referred to as secondary fluorescence.
(nuclear dyes: acridine orange, propidium iodide; mitochondria:
rhodamine 123; lipophilic dyes: eg. DiI to visualize membranes,
liposomes, etc.).
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IMMUNOFLUORESCENCE:
SIMULTANEOUS DETECTION OF MORE ANTIGENS
Actin
Microtubuli
NucleusBovine pulmonary arthery endothelial cells
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- utilizes an electron microscope for examination of tissue samples;
here the sample is exposed to a chain of electrons which enables
one to obtain higher magnification than LM and examine cellular
ultrastructure. After electrons fall on a fluorescent screen, a visible
picture is generated.
C) ELECTRON MICROSCOPY
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- Scanning EM: enables observation of a sample surface; an
electron beam moves sequentially from point to point across ametal-covered surface (of a sample) and is reflected to a detector
where the image is formed from lines (of points) as in TV.
A louse clings to a human hair.
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- Transmission EM: electrons penetrating a tissue section are
absorbed in dense structures; resulting image carries information ondistribution of electrondense structures in a section. The technique
requires ultrathin sections (40-80 nm, embedded in Epoxy resin
Durcupan-Epon; from the same tissue block, semithin sections 0.1-1.0 m can be also cut and examined in LM). To enhance contrast
of tissues containing atoms of a low molecular weight, heavy
metals (OsO4 and uranyl acetate) are used.
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- high resolution power of TEM can be also used for
evaluation of subcellular distribution of antigens
(antibodies can be labelled with electrondense markers,
eg. colloid gold). In the same way, lectins, avidin, protein
A and probes for in situ hybridization can be labelled.
- tissue samples stained for histochemistry (eg. detection
of enzymatic activity) can be also processed for TEM -
this is possible if resulting reaction product reacts with
heavy metals (positivity is then electrondense).
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D) AUTORADIOGRAPHY- extremely sensitive method that enables one to visualize deposition
of radionuclides in tissues. A radioactive isotope can be introduced
into tissues. For example, in vivo after administration of a compound
labelled with an isotope that is in the scope of biochemical reactions
incorporated into the nucleus, membrane, secretory granules, etc. A
radionuclide can be also utilized to label other molecules (antibodies,
lectins, oligonucleotides, etc.) that can be incubated with histological
sections. Sections are then covered with a photographic emulsion; in
the site of radioactivity, silver is reduced (similarly as in the process of
developing photographs).