Histochemistry - Principles and Methods Flashcards
Define histochemistry
The study of tissue-chemical interactions involved in the visualisation of cell/tissue components
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
Give two methods of staining
Direct bonding
Metal impregnation
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.
What are the five main staining methods
Staining by direct binding
Metal impregnation
Lysochrome
Histochemical
Indirect staining methods
What is meant by staining by direct binding?
Bonding, affinity for chromogen (direct and indirect)
Dye-staining
What is the metal impregnation method of staining
Metallic ion staining e.g. silver or gold ions
Staining by adsorption
What is lysochrome method of staining?
Staining by solubility
e/g/ lipid staining (with dyes)
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
What is the indirect staining method
Through antibody or oligonucleotide probe binding to substrate and linking to a chromogen (label)
Immunhistochemistry
What is a chromogen
Colouring agent/chemical
What is a substrate?
Component in tissue or cells which is stained
What is meant by acidophilic?
Bonds to acidic groups/dyes
What is meant by basophilic
Binds to basic groups/dye
What is meant by dichrome
Two colour
What is meant by trichrome
Three colour
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
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
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
