Transcript Document

Histology 1.2.: Immunohistochemistry
Immunohistochemistry uses the principle of immunity:
• During development the immune system recognizes foreign
proteins as antigens
• If foreign proteins invade the body, this evokes immune response
• One type of immune response is the production of highly specific
molecules against the foreign proteins. These are called antibodies,
binding with high affinity to the antigens
• Immunocytochemistry utilizes these antibodies for the localization
of tissue components
Production of antibodies:
1. A tissue constituent is extracted from the body of X animal (e.g. goat),
and purified
2. This material is injected into the bloodstream of Y animal (e.g. rabbit),
where it behaves as antigen and evokes immune response,
thus, production of highly specific antibodies
3. The antibody can be extracted from the blood of Y animal, purified and
characterized.
Preparation of tissues for immunohistochemistry:
1. Collection of samples (tissue blocks from experimental
animals, biopsy, smears, etc.)
Fixation:
- immersion (drop the tissue block into fixative)
- perfusion through the heart
Perfusion:
1. Deep anaesthesia (Nembutal, etc.)
2. Cannule introduced to the left ventricle
or into the aorta
3. Wash out the blood with a saline
4. Fix with paraformaldehyde and/or
glutaraldehyde
5. Removal of the wanted tissue or organ
immersion-fixed for some hours
6. Sectioning
7. Incubation of sections
An example: pre-embedding
immunohistochemical reaction:
1. Antigen (green triangle)-antibody
binding in the tissue
2. Antigen-antibody binding
between the primary antibody
and the secondary antibody
labelled with either a gold
particle, or a fluorescent dye,
or an enzyme catalysing
a chromogen reaction
The results:
Epithelial cells infected
with influensa viruses
(brown dots)
in the wall of a bronchus
in the lung
Nerve cells containing the enzyme nitrogen
monoxide synthase (DAB reaction, brown
precipitate)
B
Endothelial cell culture:
Red fluorescence: actin cytoskeleton
Green fluorescence: tubulin
Blue: DAPI staining of the nucleus
(not immune staining)
IMMUNFLUORESCENCE
GAD-GFP and NPY in fluo microscope
GAD-GFP and enk, confocal micr.
The electron microscope
Brief history:
1920: physicists discovered that accelerated electrons behave
in vacuum jut like light
- they travel in straight lines and their wavelength is about
100.000 times smaller than that of light.
- the electron beam can be manipulated with electromagnetic
field just like the light with glass lenses
1931: Ernst Ruska built the first electron microscope
The transmission electron microscope (TEM)
Electron source: triode gun
1. filament: tungsten, heated up to 2700oC: emits electron cloud
2. Wehnelt cylinder: bunches the electrons into finely focused point
3. anode: has a hole in it so that the accelerated electron beam
get through it with a speed of several 100.000 km/sec
Magnification:
with the help of electromagnetic lenses:
changing the strength of the current within the coils
changes the magnification
Image formation:
the focussed electron beam reaches the extremely thin specimen
(60-90 nm), passes through it and the image is projected
to a fluorescent screen
the specimen has to be treated with heavy metal salts in order
to get contrasty image („staining”=contrasting)
Preparation fo tissues for electron microscopy:
1. Fixation: buffered solutions of paraformaldehyde and
glutaraldehyde (immersion and perfusion)
2. Staining/contrasting with osmium tetroxide
3. Dehydration: in ascending series of ethanol (50%-100%)
Staining/contrasting with 70 % ethanol saturated with uranyl acetate
4. Intermediate solvent: propylene oxide
5. Embedding: in synthetic resins e.g. Durcupan ACM (liquid at room
temperature, polymerises at 56 oC)
6. Preparation of semithin (0.5 mm) and ultrathin (60-90 nm) sections
Staining/contrasting with lead citrate.
The ultramicrotom:
The electron micrograph
nucleus
Scanning electron microscope (SEM)
Suitable to observe the surface of tissue components
Parts of SEM:
Electron optical column (short with 3 lenses)
Specimen chamber
Works like the tv screen:
- The electron beam hits the surface of the specimen which
has to be covered with a thin layer of metal (e.g. gold)
- Secondary electrons are detected and turned into an electrical
signal.
- In the monitor electrical signal is turned into light to produce
an image.
SEM images :
Red and white blood cells
Blood clotting
Pre-embedding immunocytochemistry at
electron microscopic level:
Its steps are similar to those of light microscopic
ICC but:
- Triton X-100 detergent is not allowed to use
- Instead Triton X-100 freeze-thaw in liquid
nitrogen helps the penetration of antibodies
- The immunoreaction is carried out on 60-80 mm
vibratome sections
Further steps after the immunoreaction:
-contrasting: buffered 1 % OsO4
30-60 min
-Dehydration in ascending series of ethanol
10-10 min
(70 % ethanol is saturated with uranyl acetate)
- Intermedier solvent: propylene oxide
10 min
- Durcupan : propylene oxide 1:1
30 min
- Durcupan resin
overnight
- Mounting on glass slide in Durcupan resin
- Polimerization 56 oC-on
one day
- re-embedding for ultrathin sectioning
- Preparation of ultrathin sections (60-90 nm) in ultramicrotome
- Contrasting with lead citrate
2-10 min
- View in electron microscope
Light microscopic level
Electron microscopic level
Postembedding immunogold labelling:
- Carried out on ultrathin sections
- Secondary antibody is decorated with a colloidal gold particle
Localization of gonadotrop hormon
presynaptic membrane protein