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2.2 Cellular Pathology > Practical 4: Masson Trichrome, Elastin, Gordon and Sweet's Stains > Flashcards

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

1
Q

In your own words what is connective tissue

A

Extracellular proteins

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2
Q

Write a note on connective tissue
(3)

A

Most abundant tissue type in the body

Many functions

Many different stains can be used to detect connective tissues

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3
Q

List the six functions of connective tissues

A

Structure

Mechanical

Protection

Transport of nutrients

Metabolites

Defence against pathogenic organisms

Tissue repair

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4
Q

Name three stains used for connective tissue

A

Masson’s Trichrome

Verhoeff’s Van Gieson

Gordon and Sweets silver stain

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5
Q

What are trichromes

A

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

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6
Q

What is the principle behind any trichrome
(2)

A

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

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7
Q

Comment on the permeability of rbcs in trichrome stains

A

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

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8
Q

Comment on the staining of collagen in trichrome stains

A

Collagen stains by the largest dye

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9
Q

List the five stains used in Massons Trichrome

A

Celestine Blue

Mayer’s haematoxylin

Ponceau red

Acid fuchsin

Light green

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10
Q

What is the function of celestine blue and haematoxylin

A

They combine to form a complex which intensely stains the nuclei

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11
Q

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

A

These stains complex together to form an intensely blue nuclear stain

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12
Q

Which is the smallest stain in Massons Trichrome

A

Acid fuchsin

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13
Q

What is the medium sized die in Massons Trichrome

A

Ponceaux Red

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14
Q

What is the largest dye in Massons Trichrome

A

light green

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15
Q

What is the principle behind Massons Trichrome

A

Rbcs stained with acid fuschin

Muscle and epithelium stained with ponceaux red

Collagen stained with light green

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16
Q

What do we use to differentiate in Massons Trichrome

A

Phosphomolybdic acid

17
Q

Why do we need to differentiate in Massons Trichrome

A

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

18
Q

Describe how you differentiated the Massons Trichrome

A

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

19
Q

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

A

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

20
Q

What is Verhoeffs Van Gieson stain used for?

A

Elastin fibres

21
Q

Write a note on Verhoeffs Van Gieson stain
(2)

A

Used for elastin fibres

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

22
Q

What is the Verhoef stain component of VVG
(4)

A

An iron-haematoxylin stain

Specific for elastic fibres

Forms very strong bonds with elastin

This will also stain the nuclei

23
Q

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

A

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

24
Q

What are the two stains in Van Gieson

A

Picric acid

Acid fuchsin

25
Q

What are some top tips for Verhoeffs Van Gieson

A

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

26
Q

What colour is picric acid

A

Yellow

27
Q

Why do you need to differentiate for VVG

A

The Verhoeffs haematoxylin (haematoxylin-iron stain) will stain everything

You need to get it out of everything other than the nuclei and elastin

28
Q

What is Gordon and Sweet’s stain

A

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

29
Q

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

A

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

30
Q

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

A

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

31
Q

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

A

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

32
Q

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

A

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

33
Q

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

A

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

34
Q

How are silver ions added to your G&S

A

Ammoniacal silver solution allows silver ions to bind

35
Q

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

A

Ions are reduced using formaldehyde to become visible

36
Q

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

A

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

37
Q

What is done after adding gold to your G&S

A

Fixing with sodium thiosulphate removes any unbound ions

38
Q

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

A

Counterstain with light green

39
Q

What are some top tips for Gordon and sweets

A

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

2.2 Cellular Pathology (29 decks)

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