hi techniques 2010

7/31/2019 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).

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