Histochemistry - Principles and Methods Flashcards by Aífe Hayden

Define histochemistry

The study of tissue-chemical interactions involved in the visualisation of cell/tissue components

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What is the general aim of histochemical methods of staining?

Formation of stable, coloured compounds at site of component to be detected with clear contrast to other elements present in tissue

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Give two methods of staining

Direct bonding
Metal impregnation

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Why do we need coloured stains to create contrast?
(5)

Processed tissue and tissue sections are generally colourless

They need to be stained to observe cell and tissue detail, components and structure

Various coloured dyes and stains can be used to identify different components and structures

Many of these methods are based on chemical principles

Allows components/structures to be stained selectively. Often more than one dye or stain is applied.

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What are the five main staining methods

Staining by direct binding

Metal impregnation

Lysochrome

Histochemical

Indirect staining methods

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What is meant by staining by direct binding?

Bonding, affinity for chromogen (direct and indirect)

Dye-staining

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What is the metal impregnation method of staining

Metallic ion staining e.g. silver or gold ions

Staining by adsorption

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What is lysochrome method of staining?

Staining by solubility

e/g/ lipid staining (with dyes)

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What is meant by histochemical staining?
(4)

Staining by chemical reaction

Insoluble precipitate is formed

Due to substrate creating a chromogen/coloured compound at site of reactivity

Similar to reactions seen in a test tube

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What is the indirect staining method

Through antibody or oligonucleotide probe binding to substrate and linking to a chromogen (label)

Immunhistochemistry

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What is a chromogen

Colouring agent/chemical

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What is a substrate?

Component in tissue or cells which is stained

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What is meant by acidophilic?

Bonds to acidic groups/dyes

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What is meant by basophilic

Binds to basic groups/dye

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What is meant by dichrome

Two colour

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What is meant by trichrome

Three colour

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Write about the history of dye development
(4)

Majority of dyes now are synthetic/man-made dyes

Originally dyes were only from natural sources

Our synthetic dyes are mainly made from the petrochemical industry: derivatives of benzene

Dyes used in histology are also used in the cosmetic, clothing and food industry

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What are the three considerable properties of dyes

Biochemistry of dye -> it’s structure and colour index number

Charge in aqueous solution -> is it neutral, acidic or basic

Mode of staining -> is it progressive or regressive

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What is the colour index
(5)

A number issued to dyes

Specific to each dye

An internationally accepted numbering system

Identifies chemical structure, reactivity and common names of dye

This is used to identify dyes instead of their common names as they may have multiple

What are three other properties of dyes that should be considered?

Fluorescent
Metachromatic
Lysochrome

Write about the general biochemistry of dyes
(4)

All dyes have aromatic structure

All absorb in visible range of electromagnetic spectrum

All have chromophoric structure to enable this visibility

Most have added reactive groups (auxochromes) which aid in their ionisation in water

What is the CI number for acid fuchsin

CI 42685

What are two other names for acid fuchsin

Acid violet 19

Acid magenta

What is the biochemistry behind the acid fuchsin dye?

Triphenylmethane structure

SO3- auxochrome attached, to make acidic in aqueous solutions

What is a progressive dye

These stain one component - Timed/no overstianing - e.g. eosin or neutral red

What is a regressive stain (3)

Overstains all components, required removal of excess/weakly bound dye by differentiation Crystal violet (in gram stain) Haematoxylin

What is differentiation

Microscopic control of dye removal, breaking bonds of weakly bound dye

What are mordants and how are they used

Some dyes may require addition of reactive ions, acids to enhance their reactivity Mordants are metal ions which complex with dye

Give three examples of dyes which make use of mordants

Haematoxylin Alcian blue Celestine blue

What does fluorescent mean

Emit light under UV

What does metachromatic mean

Two colours Monomeric and polymeric

What does lysochrome mean

Hydrophobic dyes - lipid stains Dyes more soluble in tissue lipids than in solvents

Write about the reactivity of dyes

Acidic dyes -> bind to basic tissue elements Basic dyes -> bind to acidic tissue elements Neutral dyes -> stain through solubility

Give an example of an acid dye

Acid fuchsin

Give an example of a basic dye

Basic fuchsin

What is affinity-based staining

Reactive groups in tissue bind to reactive groups on dyes, metals or other reagents

How does the Trichrome stain work?

Small dye goes in and is held tight by RBC's but is washed out of collagen and muscle Medium dye fills cytoplasm/muscle/collagen but cannot enter RBC's Differentiator turns out medium dye in order of collagen/muscle/cytoplasm Collagen filled with large dye molecule

How do metallic impregnation methods work (5)

Staining by absorption Application of silver and/or gold solution to produce black insoluble metal deposits at selected reactive sites Often pre-treatment to sensitise tissue components Reduction of metal salts to the metallic state by tissue constituents Metal ions in aqueous solution are adsorbed onto reaction components and reduced to stable inert metal aggregate

What is an aregentaffin reaction

Some tissue constituents have the capacity to reduce silver ions e.g. melanin or lipofuscin

What is an argyrophil reaction? (3)

Tissue constituents which have an affinity for silver ions but cannot reduce it without an extraneous reducing substance and are termed argyrophil They require a reducer e.g. nerve fibres, reticulin, basement membrane, glycogen

What is a lysochrome stain (5)

Hydrophobic associated with lipid - elective solubility - dye more soluble in lipid than its solvent Oil red O, Sudan Black B, Nile Blue sulphate - neutral fats and phospholipids

Give three lysochrome stains

Oil red O Sudan Black B Nile Blue Sulphate

What are the six criteria for histochemical reactions

Be sensitive and specific Be able to take place on tissue sections Must not destroy the tissue The end product of the reaction must be stable, visible and insoluble in solvents used Reaction must be at site of substance Reaction must be quick to prevent substance diffusing

What are the two treatment steps in Perls Prussian Blue staining

Treat with 2% Hydrochloric acid -> liberates ferric iron from protein complexes Treat with Potassium ferrocyanide -> ions complex with ferric iron to form blue compound

What is the basis of perls prussian blue staining

Ferric iron (Fe3+) binds to ferrocyanide ions forming ferriferrocyanide (blue)

What are the two treatment steps of the periodic acid Schiff reaction?

Treat with 1% Periodic acid -> diglycol groups in hexose molecules are oxidised to dialdehydes Treat with Schiff Reagent -> dialdehydes bind and recolour basic fuchsin (magenta)

Define staining

Production of coloured chemical compound through interaction of a series of chemical solutions with reactive tissue component