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BMedSc Honours > Tissue staining > Flashcards

Tissue staining Flashcards

1
Q

What is the purpose of staining tissues?

A

Thin tissue slices are colourless.Staining (with chemical dyes or heavy metals) helps to reveal detail in these samples

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2
Q

What is another means of revealing detail in colourless samples, besides staining with chemical dyes or heavy metals?

A

Manipulate optical pathways on a light microscope (phase contrast, darkfield, differential interference contrast)

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3
Q

Why is fresh tissue a problem, and how is this problem overcome?

A

Fresh tissue is soft, decays quickly (autolysis) and cannot be sliced thinly enough to view under a microscope.Solution = tissue fixation

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4
Q

What are the steps in tissue preparation?

A
  • Fixation
  • Processing
  • Sectioning
  • Mounting
  • Staining
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5
Q

Explain the steps of tissue preparation

A

Fixation: preserves tissue morphology and prevents autolysisProcessing: dehydration, clearing, infiltration, embeddingSectioning: thin sections cut on a microtome or cryomicrotomeMounting: sections mounted onto microscope slideStaining: chemical dyes impart colour contrast

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6
Q

What is the purpose of tissue fixation?

A

Preserves tissue morphology and prevents autolysis

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7
Q

What are the types of tissue fixation?

A

Chemical and physical

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8
Q

Explain two types of chemical fixatives

A

Cross-link fixatives:aldehydes and oxidising agents – form cross-links with their targets, stabilizing tissue structure
Coagulants: EtOH, MeOH,acetic acid – coagulate/precipitate proteins. Conformation changes by disrupting hydrophobic interactions. Rarely used

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9
Q

Explain a type of physical fixation

A

Physical fixation:

Freeze in liquid N2 with cryo-protectant (isopentane or propane)

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10
Q

Name two types of cryo-protectants used in physical fixation

A

isopentane and propane

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11
Q

What are the 4 steps involved in processing?

A
  • Dehydration
  • Clearing
  • Infiltration
  • Embedding
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12
Q

What occurs during dehydration?

A

Sample placed in ethanol to remove water

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13
Q

What occurs during clearing?

A

xylene, toluene and chloroform remove the ethanol

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14
Q

What occurs during infiltration?

A

xylene is replaced with molten paraffin which infiltrates tissue and provides a hard matrix for cutting

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15
Q

What occurs during embedding?

A

Tissue is placed in a mould containing molten wax/resin. Wax/resin then hardens
Ensure tissue is orientated into the plane of preference

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16
Q

What is an important consideration during embedding?

A

Ensure tissue is orientated into the plane of preference

17
Q

What occurs during sectioning?

A

Thin 5 um sections of wax-hardened tissue are cut on a microtome

18
Q

On what device is wax-hardened tissue cut?

A

Microtome

19
Q

To what thickness are wax-hardened tissues cut?

A

5um

20
Q

What occurs during cryosectioning?

A

Tissue is embedded in support medium OCT (optimal cutting temperature), then frozen. Sections are cut on a cryostat/cryomicrotome
(can either use prior chemically fixed tissue and freeze it, or use unfixed frozen tissue)

21
Q

On what device are cryo-sectioned samples cut?

A

Cryostat/cryotome

22
Q

What occurs during mounting?

A

Tissue slices are placed in a warm water bath before being mount onto microscope slide

23
Q

What occurs during staining?

A

Chemical dyes impart colour contrast. Different dyes are used to stain different cellular components.

24
Q

What are dyes composed of?

A

A benzene molecule forms the backbone of dyes, to which a chromophore is added. (an organic molecule component which absorbs light)

25
Q

Name 4 different mechanism of dye-staining

A

Acid-base interactionsPermeability and displacementDeposition/impregnation/precipitationHistochemistry

26
Q

Provide an example of an acid-base dye stain, and explain the principles underlying it

A

H&E stain
basic (+) dye (haematoxylin) binds to acidic (-) components e.g., DNA and RNA
acidic (-) dye (eosin) binds to basic (+) components e.g., cytoplasmic proteins

27
Q

What does H&E stand for?

A

H=haematoxylin

E=eosin

28
Q

What are 3 problems with H&E staining, and what are solutions to these problems?

A

Problems

  • Can’t distinguish between different tissue components with similar charge – all display same colour
  • Fine extracellular fibres not visible. Cytoplasm and extracellular fibres show similar staining
  • Mucopolysaccharides and lipids are unstainedSolutions
  • Use trichrome stains
  • Use metal salts/deposition
  • use periodic acid-Schiff stain (PAS)
29
Q

Provide an example of a permeability and displacement stain, and explain the principles underlying it

A

Trichrome stain
Trichrome stains are acid dyes (i.e., stain basic components) with the same net charge. They stain according to relative molecular weight and use principles of permeability and displacement
The permeability of the dye depends on its relative molecular weight, as well as that of the tissue components:
-lower molecular weight dyes/diffusible dyes stain penetrate easily and stain all tissue components, including denser, compact structures
-higher molecular weight dyes/colloid dyes penetrate more permeable tissue structures such as collagen
Larger dye molecules displace smaller dye molecules from permeable structures

30
Q

What does the permeability of trichrome dyes depend on?

A
  • Relative molecular weight of dye

* Relative molecular weight of tissue components

31
Q

What does the permeability of trichrome dyes depend on?

A
  • Relative molecular weight of dye

* Relative molecular weight of tissue components

32
Q

What is another name for low molecular weight dyes, and how do they work?

A

diffusible dyes

penetrate easily and stain all tissue components, including denser, compact structures

33
Q

What is another name for high molecular weight dyes, and how do they work?

A

colloid dyes
penetrate more permeable tissue structures such as collagen
larger dye molecules displace smaller dye molecules from permeable structures

34
Q

Provide an example of a deposition/impregnation/precipitation dye and explain the principles underlying it

A

Metal salt stain e.g. reticulin stain
These bulk up fine structures and make them visible
The process is as follows:
• Tissue is treated with oxidising agent which has an affinity for silver, to enhance subsequent staining.
• Tissue is then sensitized using an iron alum solution which targets and binds to the tissue element (retic fibres)
• Silver solution impregnates the fibres by replacing the sensitizer with metal salts
• Reducing agent is added, causing metal salts to be reduced to visible metallic form at binding site

35
Q

Provide an example of a histochemistry dye and explain the principles underlying it

A

Enzyme histochemistry
Chemical reaction between tissue and colourless staining solution to form a colour compound
Can be used to visualise enzyme activity in a tissue. Cannot use chemical fixation, as this may inactivate the enzymes.
Frozen sections are incubated in solutions containing substrates for the enzyme of interest.
Active enzymes react with the substrate to form a primary reaction product which is colourless.
The enzyme cleaves hydrogen from the substrate, which then which then combines with colourless tetrazolium dye salt to form an insoluble purple-blue precipitate (formazan)marking the site of enzyme activity.

36
Q

Why can’t chemical fixation be used in enzyme histochemistry?

A

May inactivate the enzymes

37
Q

What is the name of a colourless dye salt used in enzyme histochemistry?

A

Tetrazolium dye salt

38
Q

What is the name of an insoluble purple-blue precipitate used in enzyme histochemistry?

A

formazan

39
Q

Explain the principle of the periodic acid-Schiff stain (PAS)

A

PAS reacts chemically with tissue, forming a deep magenta colour in the presence of mucopolysaccharides (not lipids).

BMedSc Honours (3 decks)

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