HISTOLOGICAL TECHNIQUES

A) LIGHT MICROSCOPY B) FLUORESCENT MICROSCOPY C) ELECTRON MICROSCOPY D) AUTORADIOGRAPHY

A) LIGHT MICROSCOPY:
- 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 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.

Cytochemistry = the science that examines location of chemical components and reactions in cells (obtained for example: from tissue cultures, 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 (colloid iron according to Hale-Mller), detection of DNA (Feulgens nuclear reaction). - Enzymatic histochemistry: detection of enzymatic activity using their reaction with substrates; the resulting (coloured) reaction product is insoluble and accumulates in the site of enzymatic activity (peroxidase, alkaline phosphatase, cholinesterase, peptidase, etc.).

- 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

Immunohistochemistry: a method that enables detection of antigens in tissues with the use of labelled specific antibodies. The reaction is highly specific and sensitive. Antibodies bind antigens with high specifity and affinity (Ka= 105-1011l/mol) and form the antigenantibody 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, polysaccharide, 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).

DIRECT METHOD

INDIRECT TWO-STEP METHOD

secondary antibody (anti-immunoglobulin) labelled with a marker

antibody with a marker

primary antibody (unlabelled)

tissue/cellular antigen

tissue antigen

AVIDIN-BIOTIN METHOD LAB

Affinity (Ka-1015 l/mol) avidin labelled with a marker secondary antibody labelled with biotin

primary antibody tissue/cellular antigen

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.

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'

chain of the DNA:

B) FLUORESCENT MICROSCOPY
- examination of tissues using a special (fluorescent) microscope. In this microscope, a sample is exposed to a light 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 be observed in a microscope.

barrier filter

emitted light (visible)

reflected light is absorbed

histological slide

FLUORESCENT MICROSCOPE (epifluorescence)

- 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.). - 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

IMMUNOFLUORESCENCE: SIMULTANEOUS DETECTION OF MORE ANTIGENS

Bovine pulmonary arthery endothelial cells

Actin Microtubuli Nucleus

C) ELECTRON MICROSCOPY
- 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.

- Scanning EM: enables observation of a sample surface; an electron beam moves sequentially from point to point across a metal-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.

- Transmission EM: electrons penetrating a tissue section are absorbed in dense structures; resulting image carries information on distribution 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 0.11.0 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.

- 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).

- 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.

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).