Special methods in

histology

639

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 electron 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

Staining

Staining 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.

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

endoplasmic reticulum

Eosin stains basic structures of cell

(acidophilic, eosinophilic) – that are

predominantly proteins, ie. cytoplasma,

mitochondrias, smooth endoplasmic

reticulum, and collagen in extracellular matrix

Haematoxylin - eosin

AZAN

Azocarmine stains nuclei

(red)

Aniline blue stains

collagen fibres and

mucus (blue)

Orange G stains

cytoplasm, muscles

(orange)

Red blood cells are

Weigert van Gieson

Weigert haematoxylin

nucleus is brown

Saturn red stains

collagen fibres and

mucus (red)

Trinitrophenol (picric

acid) stains cytoplasm

and muscles (yellow)

Acid fuchsin can be

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 cytoplasm

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.

Results of staining

grey- blackbrown to

black

Heidenhain iron

haematoxylin

Heidenhain iron

haematoxylin HIH

Reticular fibres- blackgrey-blackbrownAgNO3Silver

violetResorcin

Fuchsin

Weigert resorcin-

fuchsin

red - erythrocytesredgreenblue to

black

Haematoxylin

Acid fuchsin

Light green

Green Masson

trichrome

Red – erythocytesred yellowblue to

black

Haematoxylin

Erythrosin

saffron

Yellow Masson

trichrome

Red- erythrocytes

Blue - mucus

redblueblue to

black

Haematoxylin

Acid fuchsin

Anilin blue

Blue Masson

trichrome

Red - erythrocytes

blue- 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 red

Trinitrofenol

Weigert – van

Gieson

pinkpinkBlue to

blac

Haematoxylin

Eosin

Haematoxylin-eosin

NoticeMuscleElasticCollagenNucleusDyesStaining

Permanent slide

Water is removed from tissue after staining

Cover slip is stick by resin

Permanent slide is made

Histochemistry

It uses chemical and histochemical reaction for

the detection of elements or compounds in

situ in cells and tissues

Histochemistry

Catalytic histochemistry

Affinity histochemistry

Detection of elements or

compounds

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

Perls reaction –detection of Fe3+

Fe3+ (HCl) and

potassium

ferrocyanide.

Product of reaction is

Prussian blue

Detection of organic

compounds

Carbohydrates:

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 cell

Diagnostic: celiac disease

They 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

Conditions:

To maintain the enzymatic activity and structure of tissue and cells, we use cryostat sections

Fixation degreases or inhibits completely the enzymatic activity

pH, temperature, substrate in abundance

Activators and inhibitors

For 200 enzymes are available special procedures - protocols

Methods for visualization in

catalytic histochemistry

Precipitation

Metal salt capture methods (Cobalt, Lead, cerium)

Diazonium salt methods

Indigogenic methods

Tetrazolium salt methods

DAB- diaminobenzidine methods

Affinity histochemistry

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

Lectin histochemistry –detection of mono-, di-, tri-, or even polysaccharides in the complex molecules by bond of lectins to the saccharides

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

Immunohistochemistry

Antibody binds to specific place on protein –

epitop

Antibodies– polyclonal

monoclonal

Markers

Antibodies (lectins and probes, too) have

to be visualized by markers: Fluorochromes (FITC, rhodamine)

Biotin

Enzymes (HRP, alP) – catalytic histochemistry is

used for their visualization

Colloidal gold particles

Isotopic probes

Ferritin, digoxigenin

Immunohistochemistry

Direct reaction Ag + AB

Indirect reaction Ag + AB1 + AB2

PAP reaction

Peroxidase –

Anti-peroxidase

Signal amplification

More molecules of

enzyme mean much

product and heavier

signal

ABC reaction

Biotin is marker of

secondary antibody

It reacts with avidin

that is bound to the

enzyme

Signal is amplified

Insulin

Cytokeratins

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

Lectins

Lectins are proteins or glycoproteins that agglutinate cells and/or precipitate complex carbohydrates. The binding is highly specific. Lectins are isolated from a wide variety of natural sources including plants, fungi, bacteria and vertebrates.

Application: blood grouping

mitogenic stimulation of cells (lymphocytes)

histochemical studies

Electron microskopy -TEM

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

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

Semithin section X Ultrathin section

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

Cryofracture – freeze fracture

Frozen tissues are fractured, coated by metal dust, observed in TEM

Structure of membranes

Scanning electron microscopy SEM

It allows to demonstrate the surface of cells

It has lower reolving power than TEM