Methods His to Chemistry

8/6/2019 Methods His to Chemistry

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WHY ARE CELLS

STAINED?


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The dye & the reaction product must be

actually dissolved in the stained substance

(e.g., fat staining)

A dye may be absorbed on the surface of a

structure, or dyes may be precipitated within

the structure, simply because environmentalfactors (pH, ionic strength, temperature, etc.)

favor absorption or precipitation.

Most staining reactions involve a chemical

union between dye and stained substancethrough salt linkages, hydrogen bonds, or

others (e.g., basic or acidic compounds have

tendency to form electrostatic linkages with

ionizable radicals of tissues)

MECHANISMS OF STAINING:


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Staining with these dyes results in a predictable color

pattern based in part on the acid-base characteristics

of the tissue.

Color and color distribution are not absolutely reliable

for discrimination between tissue components.

1.An acid dye exists as an anion in solution, while a

basic dye exists as a cation.

2.Basic dyes- (toluidine blue,methylene blue, hematoxylin)

stain nucleoproteins, GAGs, acid

glycoproteins; acidic dyes

(acridine orange, eosin, fuschin)

stain mitochondria, secretory granules In the hematoxylin-eosin stain (H&E), the hematoxylin-

metal complex acts as a basic dye (blue or purple

results); with eosin, acidophilic structures appear in

shades of pink.


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3.In the Masson and Mallory

trichome stains, use ismade of dye competition.

This employs a staining

sequence involving iron

hematoxylin, acid fuchsin,and light green.

For distinguishing

cellular from extracellular

components: collagen fibersstain an intense green; black or brown nuclei;

mucus & ground substances take on varying

shades ofgreen; cytoplasm stains red;

elastic fibrils, RBC and nucleoli stain pink.


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3.Neutral stains are compounds

of an acid dye and basic dye.

For instance, acid fuchsin may

be neutralized by addition of

aqueous methyl green. The

resulting neutral product is

water insoluble, but may be kept in solution bythe presence of excess amounts of either

component.

The tissue stained with such a solution may

show affinity for the acid dye, the basic dye,and for the whole compound. Wright's Stain is

formed by the combination of partially oxidized

and demethylated methylene blue and eosin.


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4.Metachromatic Stain- certain basic dyes,

such as toluidine blue, stain nucleic acids

blue (the orthochromatic color), but sulfatedpolysaccharides purple (the metachromatic

color).

When dye molecules

bound to sulfategroups are stacked

closely together, the

dye experiences a

color shift from blueto purple. Thus, a metachromatic reaction

often indicates the presence of numerous

closely packed sulfate groups.


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5.Golgi silver stain- selective for nerve cells-

they blacken when silver is reduced. Only a

few cells stain which is useful in determiningrelations among nerve cell processes.


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6.Cajals gold method- Purkinje

cells in cerebellum


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7.Weigerts Potassium dichromate stains

lipid in myelin sheath of nerve axons-

myelinated axons and node of Ranvier


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Types of Vital Staining:1. Intravital staining staining of cells or tissues

in the living body by intravenous injection ofcertain dyes of a colloidal nature as colloidal

silver of tryphan blue; e.g., phagocytic cells of

the reticulo-endothelial system become

colored and conspicuous as they engulfparticles of the dye as foreign bodies.

2. Supravital staining staining of living tissue or

fresh materials (e.g., blood cells) immediately

after death or after removal from the bodywhere the cells to be stained are still surviving.

Dyes commonly employed are Janus green and

neutral red.


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Histochemical reactions must meet the

following criteria:

During fixation, dehydration embedding, sectioningand histochemical staining, the substance being

analyzed must not diffuse out of its original site.

Procedures must not block or inactivate reactive

components being studied.

Appropriate fixative, treatments, and embedding

media must be used such that the substance to be

identified is insoluble in it.

Stain or reaction product must be colored, opaque or

electron scattering so that it can be visualized. Method employed should be specific for substance

being studied. If method is not totally specific,

controls must be run to eliminate other possible sites

of reaction.


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1.CARBOHYDRATES- PAS reaction - glycol groups in

glucose residues of carbhydrates are oxidized by

periodic acid (HIO4), giving rise to aldehyde groupsthat react with Schiff reagent, producing purple or

magenta color.

Mucinin

goblet

cell

Mucin

inIntesti

nalmuco

sa

Glycogen

in livercells


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2.LIPIDS

revealed

with dyes

soluble in

lipids

(Sudan IVand Sudan

black

confer red

and blackcolors on

lipids)


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3.NUCLEIC ACIDS: Feulgen Reaction: Mild

hydrolysis with HCl frees the aldehyde group

of deoxyribose, which is thenreacted with the Schiff's reagent.

Reaction produces red color, is

highly specific for DNA and may be

used with a counter stain for the cytoplasm.3.IRON: Prussian blue or Perls reaction- used

to demonstrate ferric iron and ferritin. The

protein is split off by HCl,

allowing the potassiumferrocyanide to combine with

the ferric iron, which forms the

ferric ferrocyanide or Prussian blue.


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6.PROTEINS: enzymes that digest cell

components are used to analyze distribution of

cell and tissue elements

7.ENZYMES:

These techniques localize various enzymes

within cells and tissues by making use of the

enzyme activity itself. The enzyme is made to react with a specific

substrate.

The product of this reaction may itself be visible

in the microscope and thus demonstrate thepresence of the enzyme at a specific location,

or the reaction product is subsequently reacted

to form a visible secondary reaction product.


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Acid Phosphatase Gomori-Takamatsu method

Formalin-fixed tissue sections are incubated in

acid phosphatase with sodium

glycerophosphate and lead nitrate

buffered to pH 5.0

Liberated phosphate ions react

with lead nitrate to produce

colorless precipitate of lead

phosphate

The latter reacts with ammonium sulfide that

produce of lead sulfide.


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Dehydrogenase

Incubate nonfixed sections in asubstrate solution of H+ acceptor

such as tetrazole

Colored soluble compoundproduced (formazan) precipitates at

site of enzymatic activity


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Peroxidase DAB method

Oxidation of DAB produces black,

electron-dense precipitate

Sections of fixed tissue are incubated withenzyme in H2O2 & diaminoazobenzidine (DAB)

Detects peroxidase activity

Important in diagnosis of leukemias

Used also as label in lectin

histochemistry, immunocytochemistry,

& hybridization


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Methods His to Chemistry