Special methods in histology 195 SFST. SEM-řádkovací elektronový mikroskop Umožňuje zobrazení...

Special methods in histology

195 SFST

SEM-řádkovací elektronový mikroskopUmožňuje zobrazení

povrchu studovaných objektů

Má menší rozůlišovací schopnost než TEM

Sampling

Sampling of tissue and cells : From the live organism (BIOPSY) From the corpse (NECROPSY)

Fixation must be made otherwise enzymes and germs (bacterias) destroy the cells (AUTOLYSIS)

Tissue block for fixation must not exceed (be bigger than) 1cm3 ( for light microscopy)

Or 1mm3 ( for electro microscopy)

Fixation

Fixation stops the metabolic events in the cell either by denaturation (destruction) of enzymes or reduction of their activity

Physical methods: Heat (microwave oven) Freezing (in liquid nitrogen; -170oC)

Chemical methods: Immersion (into fixative) Perfusion (into vessels)

Chemical fixation

Mercury, osmium, chromium

Salts of heavy metals

Acetic acid, trichloracetic acid, picric acid

Acids

Methanol, ethanolAlcohols

Formaldehyde, glutaraldehyde

Aldehydes

Fixatives

methanol, chloroform, acetic acidMethacarn

ethanol, chloroform, acetic acidCarnoy

Mercuric chloride, potassium bichromate, natrium sulphate, acetic acid

Zenker fluid

mercuric chloride, natrium chloride,acetic acid, trichloracetic acid, formaldedyde

Susa

trinitrophenol, formaldehyde, acetic acid

Bouin fluid

Formaldehyde 4%

Embedding and cutting

Tissue have to be harden or stabilized for cutting by embedding in special medias (paraffin, celloidin).

These medias are not mixable with water therefore we remove water from the tissue by alcohols (dehydratation) and then we replete it by solvent of embedding medium (xylene, toluene, acetone), which procedure is named „clearing“

Cutting

Tissue is cut in slides of one cell layer, it means m. Tissue is translucent and „well-readable“ in this case

Devices that are used for cutting are called microtomes.

Tissue slices are put on slide. They are stretched out by heat, and stick by egg white-glycerin

Microtomes

StainingStaining facilitates to distinguish tissue and cell components

The majority of dyes are water-soluble, therefore we have to remove paraffin (dewax).

Slide is covered by cover slip after the staining. Resins (Canadian or synthetic resins) are used as glue. The slide is long lasting.

Permanent slide

Water is removed from tissue after staining Cover slip is stick by resin Permanent slide is made

Resolution

Resolving power is the smallest distance between two particles at which we are able to distinguish them as two separate objects

Resolving power for light microscopy is 0,2 m.

Magnification – 1000-1500 times Resolving power for electron microscopy

is 0,2 nm

Staining

General staining Haematoxylin - eosin Masson trichrome Weigert - van Gieson Heidenhain iron haematoxylin

Selective Weigert resorcin fuchsin Silver methods

Haematoxylin - eosin

Haematoxylin stains acidic components of cell (basophilic structures) – DNA, RNA, ie. Nucleus, nucleolus, ribosomes a rough endoplasmatic reticulum

Eosin stains basic structures of cell (acidophilic, eosinophilic) – that are predominantly proteins, ie. cytoplasma, mitochondrias, smooth endoplasmatic reticulum, and collagen in extracellular matrix

Haematoxylin - eosin

Results of staining

grey- blackbrown to black

Heidenhain ironhaematoxylin

Heidenhain iron haematoxylin HIH

Reticular fibres- blackgrey-blackbrownAgNO3Silver

violetResorcinFuchsin

Weigert resorcin-fuchsin

red - erythrocytesredgreenblue to black

HaematoxylinAcid fuchsinLight green

Green Masson trichrome

Red – erythocytesred yellowblue to black

HaematoxylinErythrosinsaffron

Yellow Masson trichrome

Red- erythrocytesBlue - mucus

redblue blue to black

HaematoxylinAcid fuchsinAnilin blue

Blue Masson trichrome

Red - erythrocytesblue- mucus

Orange – redblueredAzocarmine aniline blue Orange G

AZAN

Acid fuchsin can be used instead of Saturn red, all tissues are yellow except of collagen

yellowredBrownWeigert haematoxylin Saturn redTrinitrofenol

Weigert – van Gieson

pinkpinkBlue to blac

HaematoxylinEosin

Haematoxylin-eosin

NoticeMuscleElasticCollagenNucleusDyesStaining

AZAN

Azocarmine stains nuclei (red)

Aniline blue stains collagen fibres and mucus (blue)

Orange G stains cytoplasma, muscles (orange)

Red blood cells are red - erythrocytes

Weigert van Gieson

Weigert haematoxylin nucleus is brown

Saturn red stains collagen fibres and mucus (red)

Trinitrophenol (picric acid) stains cytoplasma and muscles (yellow)

Acid fuchsin can be used instead of Saturn red, all tissues are yellow except of collagen

Green Masson Trichrome

Hematoxylin stains nuclei blue

Light green stains collagen green

Acid fuchsin stains muscle tissue red

Weigert resorcin - fuchsin

Resorcin –fuchsin stains only elastic fibres

Elective staining for elastic fibres

Heidenhain iron haematoxylin

Heidenhain iron haematoxylin stains nucleus as well as cytoplasma gray-black.

It is used for staining of muscles; and in parasitology for detection of worms in tissue.

Silver methods

Silver stains reticular and collagen fibres in brown to black.

Silver methods are used for staining of neurons in neurohistology.

Electron microscopy TEM

Method of ultra-thin section

Sampling

Fixation (glutaraldehyde, paraformaldehyde and osmium oxide)

Embedding (epoxide, polyester and acrylate resins)

Polymeration

Cutting - thickness 50-60nm

Contrasting (osmium, uran, tungsten)

Observation

TEM

Method of negative staining

Corpuscle is surrounded by electron-dense substance – phospho-tungsten acid or uranyl acetate = dense background, particles are light

Used for detection of viruses

Scanning electron microscopy SEM

It allows to demonstrate the surface of cells

It has lower reolving power than TEM

Histochemistry

It uses chemical and histochemical reaction for the detection of elements or compounds in situ in cells and tissues

HistochemistryCatalytic histochemistryAffinity histochemistry

Detection of elements or compounds

Elements: Hg, Pb, Fe, Ca, Zn and their salts

Perls reaction –detection of Fe2+

Fe2+ (HCl) and

potassium

ferrocyanide.

Product of reaction is

Prussian blue

Detection of organic compoundsCarbohydrates:

polysaccharides (glycogen) glycoproteins and proteoglycans, glycolipids

(PAS reaction – HIO4 + Schiff reagent)

Lipids (lipid soluble dyes)

Sudan dyes:

Sudan black,

Sudan IV,

oil red

Catalytic histochemistry

It allow detection of enzymes (enzymatic activities) in tissues and cells

Used for:Research: localization of enzymes in cellDiagnostic: celiac diseaseThey serves as markers for visualization in

affinity histochemistry

Catalytic histochemistry

1. histochemical reaction

Tissue with Enzyme + Substrate = Product

2. reaction –visualisation

Coloured and insoluble compound arises from colourless product of first reaction

Affinity histochemistry

Immunohistochemistry – detection of proteins (glycoproteins) by the binding of the specific antibody to the antigen

Lectin histochemistry –detection of mono-, di-, tri-, i polysaccharides in the complex molecules by binding of lectins to the saccharides

In situ hybridization – detection of specific sequence of nucleoids in DNA or m-RNA by the binding of complementary chain of probe

Monoclonal and polyclonal antibodies

Antibody binds to specific place on protein – epitop

Antibodies– polyclonal

monoclonal

Immunohistochemistry is used for :

Diagnostic of tumors and other illnesses in pathology

The most important antigens: Intermediate filaments, CD antigens, hormones,

estrogen and progesteron receptor, melanoma antigens, S-100 protein, PSA (prostatic specific antigen),proliferation specific antigens: PCNA, p53 protein, KI-67

Research