Practical 4: Masson Trichrome, Elastin, Gordon and Sweet's Stains Flashcards
In your own words what is connective tissue
Extracellular proteins
Write a note on connective tissue
(3)
Most abundant tissue type in the body
Many functions
Many different stains can be used to detect connective tissues
List the six functions of connective tissues
Structure
Mechanical
Protection
Transport of nutrients
Metabolites
Defence against pathogenic organisms
Tissue repair
Name three stains used for connective tissue
Masson’s Trichrome
Verhoeff’s Van Gieson
Gordon and Sweets silver stain
What are trichromes
Multi-dye methods that are capable of distinguishing between tissue structures such as collagen and muscle in contrasting colours
What is the principle behind any trichrome
(2)
The dyes bind electrostatically, using a series of amnionic (acid) dyes of different molecular size and molecular structure
Different tissue elements vary in permeability/porosity and are stained a series of colours
Comment on the permeability of rbcs in trichrome stains
Rbcs are the least permeable and stain with the smallest molecule dyes
Comment on the staining of collagen in trichrome stains
Collagen stains by the largest dye
List the five stains used in Massons Trichrome
Celestine Blue
Mayer’s haematoxylin
Ponceau red
Acid fuchsin
Light green
What is the function of celestine blue and haematoxylin
They combine to form a complex which intensely stains the nuclei
What does Celestine blue and Meyer’s haematoxylin do in Massons Trichrome
These stains complex together to form an intensely blue nuclear stain
Which is the smallest stain in Massons Trichrome
Acid fuchsin
What is the medium sized die in Massons Trichrome
Ponceaux Red
What is the largest dye in Massons Trichrome
light green
What is the principle behind Massons Trichrome
Rbcs stained with acid fuschin
Muscle and epithelium stained with ponceaux red
Collagen stained with light green
What do we use to differentiate in Massons Trichrome
Phosphomolybdic acid
Why do we need to differentiate in Massons Trichrome
The collagen will pick up the red colour from the acid fuschin and ponceaux red
Describe how you differentiated the Massons Trichrome
Use phosphomolybdic acid to take the red stain out of the collagen
If overdifferentiated then the muscle and rbcs will be too pale
If underdifferentiated then the collagen will still be red
What should you make sure to do after staining with light green
(2)
Blot them using filter paper
Place slide once face down on filter paper
Counterstains tend to leak out so double check it’s still present before dehydrating to paraclear i.e. after IMS check microscopically to see if there is still some green left
What is Verhoeffs Van Gieson stain used for?
Elastin fibres
Write a note on Verhoeffs Van Gieson stain
(2)
Used for elastin fibres
Two-part combination stain: Verhoeff stain component and the Van Gieson stain component
What is the Verhoef stain component of VVG
(4)
An iron-haematoxylin stain
Specific for elastic fibres
Forms very strong bonds with elastin
This will also stain the nuclei
What is the Van Gieson stain component of VVG
(4)
A counterstain specific for collagen
Combination of two acid dyes: picric acid and acid fuchsin which bind to basic proteins in tissues
Acid fuchsin stains collagen
Picric acid is smaller and stains red blood cells and muscle
What are the two stains in Van Gieson
Picric acid
Acid fuchsin
What are some top tips for Verhoeffs Van Gieson
Don’t let the verhoeffs stain dry out -> you will have to top it up
Focus on a blood vessel to find elastin fibres - this will make the differentiation step easier
Differentiate until background is clear and restain with verhoeffs verhoeffs haematoxylin If necessary
What colour is picric acid
Yellow
Why do you need to differentiate for VVG
The Verhoeffs haematoxylin (haematoxylin-iron stain) will stain everything
You need to get it out of everything other than the nuclei and elastin
What is Gordon and Sweet’s stain
A silver impregnation method for the demonstration of the argyrophilic reticulin fibres of connective tissue
Gordon and Sweet’s is an argyrophilic reaction, what does this mean
The silver which hinds to the reticulin fibres must be reduced by a chemical agent to the visible black silver deposit
What is the principle behind Gordon and Sweet’s
(7)
The reticulin fibres must first be oxidised using potassium permanganate
The reticulin fibres are then bleached with oxalic acid to remove the potassium permanganate colour out
Fibres are then exposed to iron alum to sensitise them (fibres bind iron ions which act as reactive sites for silver ions)
Ammoniacal silver solution allows silver ions to bind
Ions are reduced using formaldehyde
Post treated with gold chloride to enhance the silver deposit through aggregation of gold ions
Fixing with sodium thiosulphate removes any unbound ions
Counterstain with light green
Why must the reticulin fibres be oxidised first in Gordon and sweets
To enhance their reactivity through creation of aldehyde groups in the tissue
How and why are the reticulin fibres bleached in Gordon and Sweet’s
The reticulin fibres are then bleached with oxalic acid to remove the potassium permanganate colour out
Why do you expose the fibres to iron in G&S
Fibres are then exposed to iron alum to sensitise them (fibres bind iron ions which act as reactive sites for silver ions)
How are silver ions added to your G&S
Ammoniacal silver solution allows silver ions to bind
How do you make your silver ions visible in G&S
Ions are reduced using formaldehyde to become visible
What is done to the fibres after reducing silver ions in G&S
Post treated with gold chloride to enhance the silver deposit through aggregation of gold ions
What is done after adding gold to your G&S
Fixing with sodium thiosulphate removes any unbound ions
What is done to your G&S after fixing with sodium thiosulphate
Counterstain with light green
What are some top tips for Gordon and sweets
If there’s no colour then the potassium permanganate or iron alum didn’t work
Blot dry your light green counter stain
Double check stain is green before dehydrating and paraclear -> if not green after alcohol then go back to water and then add light green again
