Histological Techniques

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Best resolving power in LM

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0.2 micrometer

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Most used sections in laboratory classes, to protect structures?

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Parafin-embedded sections

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Differenses in LM and TEM

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Electron instead of beams of light
Electromagnetic lenses instead of glass lenses
Very thin objects, e.g. epoxy resin sections
Electron is transmitted through specimen

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Cathode in TEM

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Tungsten (wolfram) - emits electrons

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Anode in TEM

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Metallic plate with hole in its center

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Electron reflection capacity depends on

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the molecular weight of components of the specimen. Higher weight cause higher reflection

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Will biogenic atoms have good contrast due to their weight and reflection in TEM?

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No, they are light and will not give good contrast

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What are used to increase contrast in ultra thin sections in TEM?

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Heavy metals like uranium or lead

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Thickness of sections in TEM

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50-90 nm thick. Structures down to 1 nm can be seen

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What is used to preserve structures in TEM?

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Fixation - Gluteraldehyd or osmium tetroxide

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Function of gluteraldehyd?

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Retain protein components of the cell

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Function of osmium tetroxide?

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Retain lipid components - especially phopholipids. Also a heavy metal so it plays a big role in electron deflection and contrast

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What are used as embedding agent in TEM?

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Plastic, e.g. epoxy resin. Instead of paraffin

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Functino of epoxy resin in TEM sections?

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Harder than paraffin, makes sure tissue can be cut really thin. Diamond or glass cutter

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Which type of metal does stain consist?

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Heavy metal, e.g. lead citrate

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Must fixations be removed in TEM or in LM?

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Only in LM, paraffin must be removed. Epoxy resin can stay on sections in TEM

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Theoretical and practical resolving power in TEM?

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1.0 nm practial and 0.25 nm theoretical

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Where are individual cells for histological viewing taken from?

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Haematological smaers (blood or bone marrow)
Other body fluids (cerebrospinal fluid, ascites(abdominal fluid caused by liver or heart failure), pleural fluid)

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How are cell tissues removed from body?

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In vivo = Biopsy
In autopsy = Necropsy

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1 Expiration:
2 Probationary excision:
3 Probationary puncture:
4 Curettage:

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1 Whole organ or its portion removed in surgery
2 A piece or tissue cut from organ
3 Sample is taken with a thick needle from organ (liver kidney) or bone marrow (sternal puncture)
4 Scrape

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What are the 5 steps in preparation of histological sildes?

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1. Fixation
2. Cutting tissue blocks
3. Tissue sections
4. Staining sections
5. Cover sections

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Why is fixation necessary?

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To prevent tissue from spontaneous decay = autolysis = their own enzymes causes a rotting process

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What is the most common fixative?

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10% formalin (40% formaldehyde diluted in water)

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What two ways of fixation exists?

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Immersion (dipping)
Perfusion (pumping the stuff into the body via blood vessels)

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Which size is usually tissue blocks cut in?

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1x1x0.5

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Three types of tissue sections

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Frozen section
Paraffin section
Resin section

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Frozen section, advantage and disadvantage

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A:
- Detection of lipids possible
- Enzymes and antigen remain intact
D:
- Impossible to prepare thin sections
- Can´t be stored permanently

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Paraffin embedded sections, advantage and disadvantage

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A:
- Thin sections can be prepared
- Can be stained with many methods
- Microscopic structure is well prepared
- Can be archived for decades
D:
- Shrinkage of tissue
- Inactivation of enzymes
- Lipids can´t be detected

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Process of paraffin embedding

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1. Series of ethanols up to 100% and acetone
2. Ethanol is replaced by non polar xylene or benzene (makes it translucent)
3. Placed with 56 degrees celcius paraffin
4. Hardening during cooling
5. Sectioning
6. Sections put on plates, attached with gelatin or mixture of white egg and glycerol

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Processing mineralized tissues

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1. Thin plate cut from bone or tooth
2. Held against a rotating base with abrasive surfaces
3. Sections polished after reach of required thickness
4. Mounted in solid canada balsam or methacrylate

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Nucleic (basic) stains dye:

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Acidic/basophilic structures:
Chromatin of nucleus
Ergastoplasm
Ground substance of cartilage

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Haematoxylin stain

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Nuclei dark (dark blue)

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Nuclear red stain

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Nuclei red

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Basic aniline dyes

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Stain ergastoplasm. E.g. toluidine blue.
Nissl staining in nervous tissue:

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Plasmatic (acidic) stains dye:

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Basic/acidophilic structures:
Cytoplasm
Collagen
Mucus
Fibrin
Elastic fibers

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Eosin stain

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In pink.
- Granules of leukocytes
- Acidophilic cells
- Eytrhocytes

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Picric acid

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In van Gieson stains only

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What are the most common staining method used?

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Haematoxylin and eosin (HE)

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Stains for connective tissue (CT) are:

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Collagen and hyaline stains
Elastin stains
reticular fibers stains

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Collagen and hyaline stains is:

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Acidic fuschin = Deep red
Van Gieson stain
Masson´s trichrome staining

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acidic fucshin dye color

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Deep red

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Van Gieson´s stain is a mixture of what and color what tissue what color?

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Mixture of picric acid and acidic fuschin
Dye:
Collagen = red
Muscle = yellow
Musclei with haematoxylin = dark

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Blue trichrome colors:

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Collagen with aniline blue = blue
Muscle with cytoplasmic dye = red
Nuclei with haematoxylin = dark

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Green trichrome colors:

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Collagen = green
Muscle with cytoplasmic dye = red or orange
Nuclei with haematoxylin = dark

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Elastin is stained with:

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Weigert resorcin-fuscin technique. Gives a purple-black color to elastin. Nuclei counterstained with nuclear red

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Reticular fibers are stained with:

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Black (with ammonia silver nitrate)
Nuclei with nuclear red

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Neutral lipids are stained with:

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Sudan III = Red
Sudan IV = Black
Nuclei with haematoxylin = dark

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Polysaccharides that can be stained:

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- Glycogen
- Neutral mucoplysaccharides
- Mucoproteins
- Acidic mucopolysaccharides
- Mucus (mucin)

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Glycogen is best stained with

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Carmine. Gives purple red color. Nuclei with haematoxylin = dark

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Neutral mucopolysaccharides, mucoproteins and glycogen is best stained with:

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PAS-method. (Periodic acid and Schiff´s agent). Gives purple red color. Nuclei with haematoxylin = dark

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Acidic mucopolysaccharides is best stained with:

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Colloid iron = blue
Nuclei with nuclear red = red

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Mucus (mucin) is best stained with:

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Carmine. Gives a pink color. Nuclei with haematoylin = dark

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Neurohistological staining contains:

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- Neurofibrillary staining of axons
- Myelin sheaths (phospholopides)
- Impregnation of astrocytes
- Block impregnation of Neurons

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Neurofibrillary impregnation of axons is with:

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Ammonia silvar nitrate (protargol). Gives a black dye. Nuclei with nuclei red = Red

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Myelin sheaths (phospholopides) are stained with:

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Luxol blue. GIves a blue-green nulei.
Nissl substance with Cresy Violet = red

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Impregnation of astrocytes is with:

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Auric chloride and sublimate. Gives a dark purple color

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Block impregnation of neurons is with:

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Black - Silver nitrate

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Pigment melanin is stained with:

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Haemosiderin with PEARLS reaction = Blue

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Sections covering

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Stained sections are dehydrated in increasing concentrations of ethanol and xylene. Mounted in Canada balsam or DPC