Practical 4: Masson Trichrome, Elastin, Gordon and Sweet's Stains

Question 1

In your own words what is connective tissue

Answer 1

Extracellular proteins

Question 2

Write a note on connective tissue
(3)

Answer 2

Most abundant tissue type in the body

Many functions

Many different stains can be used to detect connective tissues

Question 3

List the six functions of connective tissues

Answer 3

Structure

Mechanical

Protection

Transport of nutrients

Metabolites

Defence against pathogenic organisms

Tissue repair

Question 4

Name three stains used for connective tissue

Answer 4

Masson’s Trichrome

Verhoeff’s Van Gieson

Gordon and Sweets silver stain

Question 5

What are trichromes

Answer 5

Multi-dye methods that are capable of distinguishing between tissue structures such as collagen and muscle in contrasting colours

Question 6

What is the principle behind any trichrome
(2)

Answer 6

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

Question 7

Comment on the permeability of rbcs in trichrome stains

Answer 7

Rbcs are the least permeable and stain with the smallest molecule dyes

Question 8

Comment on the staining of collagen in trichrome stains

Answer 8

Collagen stains by the largest dye

Question 9

List the five stains used in Massons Trichrome

Answer 9

Celestine Blue

Mayer’s haematoxylin

Ponceau red

Acid fuchsin

Light green

Question 10

What is the function of celestine blue and haematoxylin

Answer 10

They combine to form a complex which intensely stains the nuclei

Question 11

What does Celestine blue and Meyer’s haematoxylin do in Massons Trichrome

Answer 11

These stains complex together to form an intensely blue nuclear stain

Question 12

Which is the smallest stain in Massons Trichrome

Answer 12

Acid fuchsin

Question 13

What is the medium sized die in Massons Trichrome

Answer 13

Ponceaux Red

Question 14

What is the largest dye in Massons Trichrome

Answer 14

light green

Question 15

What is the principle behind Massons Trichrome

Answer 15

Rbcs stained with acid fuschin

Muscle and epithelium stained with ponceaux red

Collagen stained with light green

Question 16

What do we use to differentiate in Massons Trichrome

Answer 16

Phosphomolybdic acid

Question 17

Why do we need to differentiate in Massons Trichrome

Answer 17

The collagen will pick up the red colour from the acid fuschin and ponceaux red

Question 18

Describe how you differentiated the Massons Trichrome

Answer 18

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

Question 19

What should you make sure to do after staining with light green
(2)

Answer 19

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

Question 20

What is Verhoeffs Van Gieson stain used for?

Answer 20

Elastin fibres

Question 21

Write a note on Verhoeffs Van Gieson stain
(2)

Answer 21

Used for elastin fibres

Two-part combination stain: Verhoeff stain component and the Van Gieson stain component

Question 22

What is the Verhoef stain component of VVG
(4)

Answer 22

An iron-haematoxylin stain

Specific for elastic fibres

Forms very strong bonds with elastin

This will also stain the nuclei

Question 23

What is the Van Gieson stain component of VVG
(4)

Answer 23

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

Question 24

What are the two stains in Van Gieson

Answer 24

Picric acid

Acid fuchsin

Question 25

What are some top tips for Verhoeffs Van Gieson

Answer 25

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

Question 26

What colour is picric acid

Answer 26

Yellow

Question 27

Why do you need to differentiate for VVG

Answer 27

The Verhoeffs haematoxylin (haematoxylin-iron stain) will stain everything You need to get it out of everything other than the nuclei and elastin

Question 28

What is Gordon and Sweet's stain

Answer 28

A silver impregnation method for the demonstration of the argyrophilic reticulin fibres of connective tissue

Question 29

Gordon and Sweet's is an argyrophilic reaction, what does this mean

Answer 29

The silver which hinds to the reticulin fibres must be reduced by a chemical agent to the visible black silver deposit

Question 30

What is the principle behind Gordon and Sweet's (7)

Answer 30

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

Question 31

Why must the reticulin fibres be oxidised first in Gordon and sweets

Answer 31

To enhance their reactivity through creation of aldehyde groups in the tissue

Question 32

How and why are the reticulin fibres bleached in Gordon and Sweet's

Answer 32

The reticulin fibres are then bleached with oxalic acid to remove the potassium permanganate colour out

Question 33

Why do you expose the fibres to iron in G&S

Answer 33

Fibres are then exposed to iron alum to sensitise them (fibres bind iron ions which act as reactive sites for silver ions)

Question 34

How are silver ions added to your G&S

Answer 34

Ammoniacal silver solution allows silver ions to bind

Question 35

How do you make your silver ions visible in G&S

Answer 35

Ions are reduced using formaldehyde to become visible

Question 36

What is done to the fibres after reducing silver ions in G&S

Answer 36

Post treated with gold chloride to enhance the silver deposit through aggregation of gold ions

Question 37

What is done after adding gold to your G&S

Answer 37

Fixing with sodium thiosulphate removes any unbound ions

Question 38

What is done to your G&S after fixing with sodium thiosulphate

Answer 38

Counterstain with light green

Question 39

What are some top tips for Gordon and sweets

Answer 39

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