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Techniques In Neuroscience > Immunohistochemistry: sample preparation > Flashcards

Immunohistochemistry: sample preparation Flashcards

1
Q

Why examine tissues and cells?

A

To study tissue anatomy and cytoarchitecture
Distribution of proteins
Pathological changes in disease

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2
Q

Use of histological study of tissues

A

In clinical diagnostics of neuropathology

In basic and translational research

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3
Q

Name tissue sources

A

Animal models
Post-mortem
Pathology samples
Surgical material

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4
Q

Advantages of animal models

A

Study different stages of a disease
Study effects of specific mutations
Can assess therapeutic strategies

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5
Q

Disadvantages of animal models

A

Nor gully recapitulate human disease

Ethical concerns

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6
Q

Advantages of post-mortem donor tissue, tissue samples, and surgical surplus

A

Reduce the need for animal research

Arguably better for studying human disease

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7
Q

Disadvantages of post-mortem donor tissue, tissue samples, and surgical surplus

A

Ethical concerns
Limited tissue supply
Low availability of early stages of the disease

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8
Q

Which process starts after the removal of live tissue?

A

Irreversible apoptosis and lysis

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9
Q

Which tissues are more prone to cellular damage?

A

Post-mortem tissues

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10
Q

Aims of tissue preparation

A

Preserve tissue in a life-like manner as possible

Prevent irreversible cell/tissue destruction

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11
Q

Methods of tissue preparation

A

Chemical fixation and Cryopreparation

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12
Q

Type of chemical fixative which preserves morphology better but staining is poor

A

Glutaraldehyde

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13
Q

Type of chemical fixative which has intermediate preservation of morphology and staining

A

Formaldehyde

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14
Q

Types of chemical fixatives in which staining is better but has poor morphology preservation

A

Acetic acid and methanol

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15
Q

Use of chemical fixatives

A

Stabilize proteins and other macromolecules

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16
Q

Characteristics of glutaraldehyde and formaldehyde

A 
Fixation process slow
Cross-linking fixatives
Anchor proteins
Create covalent bonds between proteins in tissue
Preserve protein tertiary structure
17
Q

Characteristics of ethanol and ethanol

A

Disrupt hydrophobic bonds between proteins
Irreversibly precipitation
Less suitable to antibody-based techniques

18
Q

What is immersion fixation?

A

Fresh tissue is immersed in fixing fluid and gently agitated

Preferred for small dissected samples
Large samples less agitation

19
Q

What is perfusion fixation?

A

Injection of fixing fluid in animal

The preferred method when possible

20
Q

Factors that affect the quality of fixation

A

pH changes
Length of incubation (1mm/hr preferred)
Sample size (<5mm3 preferred)
TEmperature (4ºC less penetration) (room temperature less degeneration)

21
Q

What is cryopreservation?

A

Preservation of tissue structure and components by freezing them rapidly without fixation

22
Q

Name methods of cryopreservation

A

Snap-freezing the sample with dry ice or liquid nitrogen

23
Q

Advantages of cryopreservation

A 
Fastest method
Minimal changes to protein structure
Rapid cooling (-70ºC liquid nitrogen) minimizes the damage of tissue (ice-crystal artifact)
24
Q

Disadvantages of cryopreservation

A

Poor morphology
Degradation continues
Requires special cold storage equipment
Do not permanently fixate tissue

25
Q

What is the aim of embedding?

A

Provide a solid medium that will give support for tissue structure and sufficient rigidity to enable cutting of thin sections

26
Q

Which embedding method is the more common?

A

Paraffin wax

27
Q

Name embedding method from hard to soft

A

Plastic resins
Paraffin wax
Gelatine Agarose

28
Q

Characteristics of paraffin wax

A

Hard to support tissue
Soft enough to cut
Not soluble in water
Sample needs to be processed

29
Q

Name stages of the Paraffin wax process.

A

Dehydration in alcohol 70%-90%-100%
Clearing. Use a solvent like a xylene
Infiltration with molten paraffin wax (replacing the xylene)
Embedding. Oriented in a metal mold containing molten paraffin was and allow to cool 4ºC
Remove from metal mold ready to microtomy/storage

30
Q

How do we embed tissues infiltrated with paraffin wax?

A

Transfer to a molten wax tray on an embedding station

Placed in metal molds filled with molten wax Oriented before placing the cold plate to set the wax

31
Q

Why do we use a cassette in the embedding process?

A

To provide support after removed from the metal mold

32
Q

Name some uses of plastic cassettes

A

Support in the embedding process
Support in sectioning process
Record type, date and time of tissue sample

33
Q

What is sectioning?

A

Process of cutting thin slices from the sample with a microtome for microscopic examination

Techniques In Neuroscience (9 decks)

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