Histology 1.2.: ImmunohistochemistryHistology 1.2.: Immunohistochemistry

Immunohistochemistry uses the principle of immunity:Immunohistochemistry uses the principle of immunity:

• During development the immune system recognizes foreign During development the immune system recognizes foreign proteins as antigensproteins as antigens

• If foreign proteins invade the body, this evokes immune responseIf foreign proteins invade the body, this evokes immune response• One type of immune response is the production of highly specific One type of immune response is the production of highly specific

molecules against the foreign proteins. These are called antibodies, molecules against the foreign proteins. These are called antibodies, binding with high affinity to the antigensbinding with high affinity to the antigens

• Immunocytochemistry utilizes these antibodies for the localization Immunocytochemistry utilizes these antibodies for the localization of tissue componentsof tissue components

Production of antibodies:Production of antibodies:1.1. A tissue constituent is extracted from the body of X animal (e.g. goat), A tissue constituent is extracted from the body of X animal (e.g. goat),

and purifiedand purified2. This material is injected into the bloodstream of Y animal (e.g. rabbit), 2. This material is injected into the bloodstream of Y animal (e.g. rabbit),

where it behaves as antigen and evokes immune response, where it behaves as antigen and evokes immune response, thus, production of highly specific antibodiesthus, production of highly specific antibodies

3. The antibody can be extracted from the blood of Y animal, purified and 3. The antibody can be extracted from the blood of Y animal, purified and characterized.characterized.

Preparation of tissues for immunohistochemistry:Preparation of tissues for immunohistochemistry:

1.1. Collection of samples (tissue blocks from experimental Collection of samples (tissue blocks from experimental animals, biopsy, smears, etc.)animals, biopsy, smears, etc.)

Fixation:Fixation: - immersion (drop the tissue block into fixative)- immersion (drop the tissue block into fixative)- perfusion through the heart- perfusion through the heart

Perfusion: Perfusion: 1.1. Deep anaesthesia (Nembutal, etc.)Deep anaesthesia (Nembutal, etc.)2.2. Cannule introduced to the left ventricle Cannule introduced to the left ventricle

or into the aortaor into the aorta3.3. Wash out the blood with a salineWash out the blood with a saline4.4. Fix with paraformaldehyde and/orFix with paraformaldehyde and/or

glutaraldehydeglutaraldehyde5. Removal of the wanted tissue or organ5. Removal of the wanted tissue or organ

immersion-fixed for some hoursimmersion-fixed for some hours6. Sectioning6. Sectioning7. Incubation of sections7. Incubation of sections

An example: pre-embedding An example: pre-embedding immunohistochemical reaction:immunohistochemical reaction:

1.1. Antigen (green triangle)-antibodyAntigen (green triangle)-antibodybinding in the tissuebinding in the tissue

2. Antigen-antibody binding 2. Antigen-antibody binding between the primary antibody between the primary antibody and the secondary antibody and the secondary antibody labelled with either a gold labelled with either a gold particle, or a fluorescent dye, particle, or a fluorescent dye, or an enzyme catalysing or an enzyme catalysing a chromogen reactiona chromogen reaction

The results:The results:

Epithelial cells infected Epithelial cells infected with influensa viruses with influensa viruses (brown dots)(brown dots)in the wall of a bronchus in the wall of a bronchus in the lungin the lung

BB

Nerve cells containing the enzyme nitrogen Nerve cells containing the enzyme nitrogen monoxide synthase (DAB reaction, brown monoxide synthase (DAB reaction, brown precipitate)precipitate)

Endothelial cell culture:Endothelial cell culture:Red fluorescence: actin cytoskeletonRed fluorescence: actin cytoskeletonGreen fluorescence: tubulinGreen fluorescence: tubulinBlue: DAPI staining of the nucleusBlue: DAPI staining of the nucleus (not immune staining)(not immune staining)

IMMUNFLUORESCENCEIMMUNFLUORESCENCE

GAD-GFP and NPY in fluo microscopeGAD-GFP and NPY in fluo microscope GAD-GFP and enk, confocal micr. GAD-GFP and enk, confocal micr.

The electron microscopeThe electron microscope

Brief history:Brief history:1920:1920: physicists discovered that accelerated electrons behave physicists discovered that accelerated electrons behave

in vacuum jut like light in vacuum jut like light - they travel in straight lines and their wavelength is about - they travel in straight lines and their wavelength is about 100.000 times smaller than that of light.100.000 times smaller than that of light.- the electron beam can be manipulated with electromagnetic- the electron beam can be manipulated with electromagnetic field just like the light with glass lensesfield just like the light with glass lenses

1931:1931: Ernst Ruska built the first electron microscopeErnst Ruska built the first electron microscope

The transmission electron microscope (TEM)The transmission electron microscope (TEM)

Electron source: triode gunElectron source: triode gun1. filament: tungsten, heated up to 27001. filament: tungsten, heated up to 2700ooC: emits electron cloudC: emits electron cloud2. Wehnelt cylinder: bunches the electrons into finely focused point2. Wehnelt cylinder: bunches the electrons into finely focused point3. anode: has a hole in it so that the accelerated electron beam 3. anode: has a hole in it so that the accelerated electron beam

get through it with a speed of several 100.000 km/secget through it with a speed of several 100.000 km/sec

Magnification:Magnification:

with the help of electromagnetic lenses: with the help of electromagnetic lenses: changing the strength of the current within the coils changing the strength of the current within the coils changes the magnificationchanges the magnification

Image formation:Image formation:the focussed electron beam reaches the extremely thin specimenthe focussed electron beam reaches the extremely thin specimen(60-90 nm), passes through it and the image is projected(60-90 nm), passes through it and the image is projectedto a fluorescent screento a fluorescent screenthe specimen has to be treated with heavy metal salts in order the specimen has to be treated with heavy metal salts in order to get contrasty image („staining”=contrasting)to get contrasty image („staining”=contrasting)

Preparation fo tissues for electron microscopy:Preparation fo tissues for electron microscopy:

1.1. Fixation: buffered solutions of paraformaldehyde and Fixation: buffered solutions of paraformaldehyde and glutaraldehyde (immersion and perfusion)glutaraldehyde (immersion and perfusion)

2. Staining/contrasting with osmium tetroxide2. Staining/contrasting with osmium tetroxide

3. Dehydration: in ascending series of ethanol (50%-100%)3. Dehydration: in ascending series of ethanol (50%-100%)Staining/contrasting with 70 % ethanol saturated with uranyl acetateStaining/contrasting with 70 % ethanol saturated with uranyl acetate

4. Intermediate solvent: propylene oxide4. Intermediate solvent: propylene oxide

5. Embedding: in synthetic resins e.g. Durcupan ACM (liquid at room 5. Embedding: in synthetic resins e.g. Durcupan ACM (liquid at room temperature, polymerises at 56 temperature, polymerises at 56 ooC)C)

6. Preparation of semithin (0.5 6. Preparation of semithin (0.5 m) and ultrathin (60-90 nm) sectionsm) and ultrathin (60-90 nm) sections Staining/contrasting with lead citrate.Staining/contrasting with lead citrate.

The ultramicrotom:The ultramicrotom:

The electron micrographThe electron micrograph

nucleusnucleus

Scanning electron microscope (SEM)Scanning electron microscope (SEM)

Suitable to observe the surface of tissue componentsSuitable to observe the surface of tissue componentsParts of SEM:Parts of SEM:

Electron optical column (short with 3 lenses)Electron optical column (short with 3 lenses)Specimen chamberSpecimen chamber

Works like the tv screen:Works like the tv screen:- The electron beam hits the surface of the specimen which - The electron beam hits the surface of the specimen which has to be covered with a thin layer of metal (e.g. gold)has to be covered with a thin layer of metal (e.g. gold)- Secondary electrons are detected and turned into an electrical Secondary electrons are detected and turned into an electrical signal.signal.- In the monitor electrical signal is turned into light to produceIn the monitor electrical signal is turned into light to produce an image.an image.

SEM images :SEM images :

Red and white blood cellsRed and white blood cells

Blood clottingBlood clotting

Pre-embedding immunocytochemistry at Pre-embedding immunocytochemistry at electron microscopic level:electron microscopic level:

Its steps are similar to those of light microscopicIts steps are similar to those of light microscopicICC but:ICC but:- Triton X-100 detergent is not allowed to use- Triton X-100 detergent is not allowed to use- Instead Triton X-100 freeze-thaw in liquid - Instead Triton X-100 freeze-thaw in liquid nitrogen helps the penetration of antibodiesnitrogen helps the penetration of antibodies- The immunoreaction is carried out on 60-80 - The immunoreaction is carried out on 60-80 m m vibratome sectionsvibratome sections

Further steps after the immunoreaction:Further steps after the immunoreaction:

-contrasting: buffered 1 % OsO4contrasting: buffered 1 % OsO4 30-60 min30-60 min-Dehydration in ascending series of ethanolDehydration in ascending series of ethanol 10-10 min10-10 min

(70 % ethanol is saturated with uranyl acetate)(70 % ethanol is saturated with uranyl acetate)- Intermedier solvent: propylene oxideIntermedier solvent: propylene oxide 10 min10 min- Durcupan : propylene oxide 1:1 Durcupan : propylene oxide 1:1 30 min30 min- Durcupan resin- Durcupan resin overnightovernight- Mounting on glass slide in Durcupan resin- Mounting on glass slide in Durcupan resin- Polimerization 56 - Polimerization 56 ooC-onC-on one dayone day- re-embedding for ultrathin sectioning- re-embedding for ultrathin sectioning- Preparation of ultrathin sections (60-90 nm) in ultramicrotome- Preparation of ultrathin sections (60-90 nm) in ultramicrotome- Contrasting with lead citrate- Contrasting with lead citrate 2-10 min2-10 min- View in electron microscope- View in electron microscope

Light microscopic levelLight microscopic level Electron microscopic levelElectron microscopic level

Postembedding immunogold labelling:Postembedding immunogold labelling:- Carried out on ultrathin sections- Carried out on ultrathin sections- Secondary antibody is decorated with a colloidal gold particle- Secondary antibody is decorated with a colloidal gold particle

Localization of gonadotrop hormonLocalization of gonadotrop hormon presynaptic membrane proteinpresynaptic membrane protein