Fixation and staining techniques (Lecture 1 and 2) Flashcards
Explain electron microscopy.
Electron microscope:
- The beam of electrons interacts with atoms in the tissue or atoms of heavy metals bound to the tissue (lead, osmium, uranium).
- Shorter wavelength -
- Theoretical resolution 0.2 nm
- Biological samples not better than 1.5 nm
What are the steps in the Hematoxylin-eosin staining procedure?
I. Deparaffinization
II. Staining
III. Dehydrating
IV. Clearing
V. Mounting (cover slipping)
What kind of staining is used here?
What kind of staining is used here?
Weigert van Gieson
What kind of staining is used here?
Weigert van Gieson
What kind of staining is used here?
Weigert van Gieson
What kind of staining is used here and what is the slide of?
Masson‘s green trichrome
Trachea
What stain is used here?
Masson‘s green trichrome.
Which stain is used here?
Masson‘s green trichrome.
Which stain is used here?
Masson‘s yellow trichrome.
Which stain is used here?
AZAN
Which stain is used here?
AZAN
Which stain is used here?
AZAN:
Nuclei and muscles – red
Connective tissue - blue
Which stain is used here?
Heidenhain‘s haematoxylin - special staining methods.
Which staining method is used here?
Silver impregnation (reticular fibres).
Which staining method is used here?
Silver impregnation (reticular fibres)
Which staining method is used here?
Semithin Epon section – toluidine blue
Explain why fixation is used and what its requirements are.
What is physical fixation?
Heat, microwave, freezing
What is chemical fixation?
Submerging samples in the fluid fixative (immersion fixation).
Perfusion by the fixative (perfusion-fixation in an experimental use).
Some fixatives promote cross-linking of proteins (formaldehyde, glutaraldehyde).
The other precipitation of proteins (picric acid, mercuric dichloride).
What are the different types of fixatives?
How are tissues processed?
Explain how parafin wax is used in embedding.
Explain how microtomes are used in histology
Explain the components of a light microscope.
LIGHT MICROSCOPE
- Stained sections are examined by transillumination;
- Resolving power: 0.2 μm
Mechanical components:
- Base, body tube (focus adjusting screws)
- Object stage - with aperture, through which the light may pass support the glass bearing the specimen
Source of light:
- Electric lamp (low voltage)
- Controlled movement in two directions
- Substage condensor - vertical adjustment
- Collects and focuses light into the plane of object
- Aperture/iris diaphragm (adjustment of the light intensity)
Optical components:
Objective lenses (objectives):
- Gather light that has passed through specimen
- Magnify real image
Objectives are mounted onto a revolving nosepiece which enable to change of objectives of the various magnifying power:
4x (red line)
10x (yellow line )
40x (blue line)
100x (white, ol.im.)
Resolving power = Ability to resolve detail (indicated by objective numerical aperture).
Ocular lens (eye piece):
Binocular microscope = A pair of the ocular lenses - further magnification of image.
- Projects image onto the viewer ́s retina.
Final megnification = objective lens x ocular lens magnifications.
Explain how a binocular microscope is used.
- Switching on the light.
- Adjustment of the visual field - adjustment of the interpupillary.
Distance (number of this distance found on the graduated scale must be set onto scale of the right ocular by rotation of the scale line). - Fixing of glass slide onto object stage - specimen must be placed in area of the aperture, cover glass must be upwards.
- Approaching of slide to objective lens by movement of the coarse adjustment screw (working distance is settled on a microscope).
- Focusing of both ocular lenses.
RIGHT OCULAR:
Focusing by rotation of the fine/micrometrical screw.
Left eye is closed and number of the interpupillary distance is set onto scale.
LEFT OCULAR:
Right eye is closed - focusing by rotation of the ocular.
Compare and contrast electron and light microscopy (scheme).
How must a sample be processed for electron microscopy?
Fixation
Embedding
Sectioning
Contrasting
What is histochemistry and how can DNA, RNA, lipids and polysaccharides be detected using this technique?
Histochemical methods:
- Based on the specific chemical reactions, producing insoluble coloured or electron dense reaction products.
- Used for detection of various chemical substances or expresseion of enzyme activity in tissue sections.
DNA - Feulgen’s reaction
RNA - Methylene or toluidine blue (control test with RNA-ase)
Lipids - Suban dyes, oil red (fat), fast luxol blue MBS (phospholipids - myelin sheath).
Polysaccharides:
PAS reaction –> glycogen, mucopolysaccharides (GAG), glycoproteins, glycolipids.
Alcian blue –> acid mucopolysaccharides (mucus, heparin granules).
Explain how polysaccharides can be seen using histochemical techniques.
Polysaccharides in human organism occur as:
- Glycogen (hepatocytes, cardiomyocytes)
- Mucopolysaccharides/glycosaminglycans (in the mucus, ground substance of the extracellular matrix),
- Glycoproteins, glycolipids.
They can be demonstrated by PAS reaction.
This histochemical method is based on the ability of Schiff ́s reagent (bleached basic fuchsin) to react with aldehyde groups to give distinctive purple (red, magenta) colour.
Aldehyde groups of carbohydrates are revealed by oxidation with periodic acid.
Alcian blue is a special dye for staining GAGs in mucous and goblet cells.
Which histochemical techniquie is used here?
Alcian Blue
Which histochemical techniquie is used here?
PAS
Which histochemical techniquie is used here?
PAS
Which histochemical techniquie is used here?
Oil red
Which histochemical techniquie is used here?
Brain - Luxol blue + nuclear red
White matter (myelin) - blue
Explain how the activity of alkaline phosphatase can be detected.
Brush border of the absorptive epithelium of proximal tubules in kidney.
Azocoupling method:
Incubation solution - alpha naphtol
- Phosphate, Fast Red TR, buffer pH 9
Principle of azocoupling method:
- Enzyme splits substrate into phosphate and naphtol.
- Released naphtol is coupled with diazonium salt to form an insoluble colored azo-dye.
Which histochemical techniquie has been used here?
Alkaline phosphatase
Azocoupling reaction
Kidney
Which histochemical technique has been used here?
Alkaline phosphatase
Azocoupling reaction
Kidney
Which histochemical technique has been used here?
Non-specific esterase
Kidney
Which histochemical technique has been used here?
On the left:
- Gallbladder
- PAS reaction
- Mucus and reticular fibers = purple
On the right:
- Gallbladder
- Alcian blue + nuclear red
- Mucus = blue
- Nuclei = red
What is immunohistochemistry?
Methods based on the specifity of the reaction between ANTIGEN and ANTIBODY are used for detection of specific proteins and certain macromolecules.
Antibodies are labelled by coupling with:
- Fluorescent compound (examination in fluorescence microscope).
- Enzyme: peroxidase, alkaline phosphatase (detection by histochemical method, examination in light microscope).
- Gold particles (examination in electron microscope)
Direct method
- Labelled antibody binds to antigen.
Indirect methods:
- Non-labelled antibody (primary) is bound to antigen.
- Secondary labelled antibody binds to primary antibody. This method is more sensitive.
Which immunohistochemical technique is used here?
Immunoperoxidase reaction
Which immunohistochemical technique is used here?
Alkaline phosphatase as an enzymatic label
Which immunohistochemical technique is used here?
Immunofluorescent detection
