Methods in histology

Microscopy 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

Observation of the live cells

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) Immersion (into fixative) Perfusion (into vessels)

Chemical methods:

Chemical fixation
Aldehydes
Alcohols Acids Salts of heavy metals

Formaldehyde, glutaraldehyde
Methanol, ethanol Acetic acid, trichloracetic acid, picric acid Mercury, osmium, chromium

Fixatives
Formaldehyde 4%

Bouin fluid
Susa

trinitrophenol, formaldehyde, acetic acid

mercuric chloride, natrium chloride,acetic acid, trichloracetic acid, formaldedyde Mercuric chloride, potassium bichromate, natrium sulphate, acetic acid ethanol, chloroform, acetic acid methanol, chloroform, acetic acid

Zenker fluid

Carnoy Methacarn

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 isnamedclearing

Embedding
Tissue can be proceed in beakers in thermostat (in small laboratory) Automatic embedding machines serve for the pathologic department running

Embedding

Cutting

Tissue is cut in slides of one cell layer, it means m. Tissue is translucent and well-readableinthiscase 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 whiteglycerin

Microtomes

Knives and blades for 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

For staining we use jars or automatic machines.

Autostainer

Permanent slide

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

Procedure
Paraffin slides Fixation Washing Dehydratation by alcohols Clearing by solvents Cryostat slides Freezing at 170 oC

Embedding in paraffin
Cutting Sticking on slide Dewaxing and rehydratation Cutting in cryostat Sticking on slide Sometime short fixation Histochemical reactions predominantly
Sometime dehydratation and clearing

Washing
Staining, histochemical reaction Dehydratation and clearing Resins

Resins or glycerin -gelatine

Break

Break

Microscope

Stative

Adjustment knob

Optic system:

Oculars Objectives Condensor Light

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

Staining

General staining

Haematoxylin - eosin Masson trichrome Weigert - van Gieson Heidenhain iron haematoxylin Weigert resorcin fuchsin Silver methods

Selective

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

Nalepeneznapodlonsklo

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
Staining Haematoxylin-eosin Weigert van Gieson Dyes Haematoxylin Eosin Weigert haematoxylin Saturn red Trinitrofenol Azocarmine aniline blue Orange G Haematoxylin Acid fuchsin Anilin blue Haematoxylin Erythrosin saffron Haematoxylin Acid fuchsin Light green Resorcin Fuchsin AgNO3 Heidenhain iron haematoxylin brown to black brown Nucleus Blue to blac Brown Collagen pink red Elastic Muscle pink yellow Acid fuchsin can be used instead of Saturn red, all tissues are yellow except of collagen Notice

AZAN

red

blue

Orange red

Red - erythrocytes blue- mucus Red- erythrocytes Blue - mucus

Blue Masson trichrome

blue to black

blue

red

Yellow Masson trichrome Green Masson trichrome Weigert resorcinfuchsin Silver Heidenhain iron haematoxylin HIH

blue to black blue to black

yellow

red

Red erythocytes

green

red

red - erythrocytes

violet grey-black grey- black Reticular fibres- black

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)