Methods in histologyMicroscopyMaňáková

2009

Content of practices

Organization of the practices

How microscopic slides are made Microscope Staining Observation of slides

What you need?

The basic knowledge is needed Pen and paper Textbook needed for histology: Junqueira, Carneiro: Basic Histology Moore,Persaud: Before we are born or Human Embryology

Observation of the live cells

Unicellular organisms

Metazoas: germ cells, blood cells, cells in tissue cultures

Observation is possible by special microscope (phase contrast microscopy) or using supravital staining

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)

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“

Embedding

Tissue can be proceed in beakers in thermostat (in small laboratory)

Automatic embedding machines serve for the

pathologic department running

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

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

Procedure

Resins or glycerin -gelatineResins

Sometime dehydratation and clearingDehydratation and clearing

Histochemical reactions predominantly

Staining, histochemical reaction

Washing

Sometime short fixationDewaxing and rehydratation

Sticking on slideSticking on slide

Cutting in cryostatCutting

Embedding in paraffin

Clearing by solvents

Dehydratation by alcohols

Washing

Freezing at – 170 oCFixation

Cryostat slidesParaffin slides

Microscope

Stative Adjustment knob

Optic system: Oculars Objectives Condensor Light

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

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

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

AZAN

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

Weigert - van Gieson

Nalepení řezů na podložní sklo

Weigert - van Gieson

Haematoxylin - eosin

Weigert-van Gieson

Green Masson Trichrome

Hematoxylin stains nuclei blue Light green stains collagen green Acid fuchsin stains muscle tissue red

Green Masson trichrome

AZAN

Yellow Masson trichrome

Haematoxylin stains nucleus blue to black

Erythrosin stains cytoplasma and muscles red

Saffron stains collagen fibres yellow

Red blood cells are red

Yellow Masson trichrome

Weigert resorcin - fuchsin

Resorcin –fuchsin stains only elastic fibres

Elective staining for elastic fibres

Weigert resorcin - fuchsin

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.

Heidenhain iron haematoxylin

Silver methods

Silver stains reticular and collagen fibres in brown to black.

Silver methods are used for staining of neurons in neurohistology.

Silver method

Cresyl violet

Cresyl violet stains DNA and RNA, ei. nucleus, nucleolus and rough endoplasmatic reticulum

Staining is used in neurohistology

Cresyl violet

Cresyl violet

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

What is necessary to know

What is fixation? Why it is performed? How we make slide? Overview. Basic methods for staining. Why we stain

tissues by various staining methods? Haematoxylin –eosin staining Light microscopy resolution (0,2 m)