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Techniques in Neuroscience > Introduction to immunohistochemistry > Flashcards

Introduction to immunohistochemistry Flashcards

1
Q

What do histological techniques allow us to study?

A

> Tissue anatomy and cytoarchitecture
Distribution of proteins
Pathological changes associated with disease

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2
Q

In which fields is the histological study of tissues essential in?

A

> Clinical diagnostic neuropathology

> Basic and translational neuroscience research

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3
Q

What are the two sources of tissue for histological studies?

A
  1. Animal models

2. Human: post-mortem donor tissue, pathology samples, surgical surplus

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4
Q

What are the advantages of animal models?

A

> Can study different stages of disease
Can study effects of specific mutations
Can access therapeutic strategies

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5
Q

What are the limitations of animal models?

A

> May not fully recapitulate human disease

> Ethical concerns

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6
Q

What are the advantages of human tissues for histological studies?

A

> Reduce need for animal research

> Arguably better for studying human disease

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7
Q

What are the limitations of human tissues for histological studies?

A

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

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8
Q

What happens to the tissue when removed from living organism or deceased donor?

A

Irreversible processes of autolysis (self-destruction) and necrosis begin
-> cellular damage

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9
Q

What is the difference between tissue from post-mortem donor and living donor?

A

Tissue from post-mortem donor more likely to have signs of cellular damage than samples from living donors

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10
Q

What are the two aims of tissue preservation for histology?

A
  1. To preserve the tissue in as life-like manner as possible

2. To prevent irreversible cell/tissue destruction

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11
Q

What are the common methods of tissue preservation?

A

> Chemical fixation

> Cryopreservation

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12
Q

What are the various chemical fixatives?

A
  • Acetic acid
  • Formaldehyde
  • Ethanol
  • Glutaraldehyde
  • Methanol
  • Picric acid
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13
Q

What does the choice of chemical fixative for preservation of tissue depend on?

A

The requirements from the experimental design

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14
Q

Why not use the chemical fixatives glutaraldehyde, acetic acid or methanol for tissue preservation?

A

> Glutaraldehyde -> best morphology, poor staining

> Acetic acid, methanol -> poor morphology, best staining

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15
Q

Why is formaldehyde the most common chemical fixative used to preserve tissue?

A

Offers best balance between morphology and staining quality

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16
Q

What are the effects of chemical fixatives?

A

Stabilise proteins and other macromolecules

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17
Q

What are the 2 categories of chemical fixatives?

A
  1. Cross linking fixatives

2. Precipitating fixatives

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18
Q

Which chemical fixatives are cross linking fixatives? What is their process of tissue preservation?

A

Formaldehyde and glutaraldehyde:
- create covalent bonds between proteins in the tissue

-> good preservation of morphology BUT slow fixation process

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19
Q

What are aldehydes and what are their action on cells?

A

Organic compounds that anchor proteins relative to each other inside the cells and between cells

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20
Q

Which chemical fixatives are precipitating fixatives? What is their process of tissue preservation?

A

Ethanol and methanol:
- disrupt hydrophobic bonds between proteins causing them to irreversibly precipitate

-> less suitable for antibody-based techniques: immunohistochemistry

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21
Q

What is a protein tertiary structure?

A

Three-dimensional structure of a protein

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22
Q

What are hydrophobic bonds?

A

They arise from the interaction of the hydrophobic amino acids with water

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23
Q

What are the methods of tissue fixation?

A
  1. Immersion fixation

2. Perfusion fixation

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24
Q

What is the method of immersion fixation?

A

Fresh tissue is placed in fixing fluid and gently agitated

  • diffuses the tissue over time
  • > not applicable to small dissected samples
  • > good to fix large samples
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25
Q

What is the method of perfusion fixation?

A

Injection of fixing fluid into the circulatory system

  • requires intact circulatory system
  • can deliver the fixative very quickly through organ or entire animal, via blood vessels
  • gives superior preservation
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26
Q

What are the factors affecting the quality of tissue fixation?

A

> Changes in pH (acidity)
Length of incubation in fixative
Specimen size (no larger than 5mm3 for optimal fixation)
Temperature

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27
Q

How does temperature affect the quality of tissue fixation?

A

> Fixation at 4°C retard degenerative changes but also reduce the penetration rate of fixative

> Room temperature fixation accelerates fixative penetration but also degenerative changes

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28
Q

What does cryopreservation of tissue consist of?

A

Preservation of tissue structure and components by freezing them rapidly without fixation
- snap-freezing the sample with dry ice or liquid nitrogen

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29
Q

What are the advantages of cryopreservation?

A

> Fastest method
Minimal changes to protein structure
Rapid cooling (-70°C using liquid nitrogen) minimises damage to the tissue (= ice-crystal artefact)

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30
Q

How does the rapid cooling with liquid nitrogen in cryopreservation minimises tissue damages?

A

-70°C using liquid nitrogen:

liquid water is converted to vitreous water without going through crystalline phase

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31
Q

What are the disadvantages of cryopreservation?

A

> Relatively poor morphology is preserved
Degradation continues over time (especially with incorrect snap-freezing process)
Requires specialist cold storage equipment (-80°)
- to keep tissue from further degradation

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32
Q

Why are tissue samples embedded in a solid medium?

A

Gives support for tissue structure, providing sufficient rigidity to enable cutting of thin sections
- not possible with cryopreserved tissue which is too hard to allow sectionning

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33
Q

Why is paraffin wax the most common embedding media of tissue samples?

A

Versatile embedding media

  • forms the metric in and around the sample
  • > prevents tissue distorsion
  • sufficiently hard to support tissues
  • soft enough to allow sections of different thickness to be cut, using microtome blade
  • not a soluble with water - tissues need to be processed before it can be embedded in paraffin wax
  • 56-60°C melting point is used
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34
Q

What are the 4 phases for processing fixed tissue to paraffin wax?

A
  1. Dehydrating
    - sequential immersions in alcohol
  2. Clearing
    - remove alcohol in solvent that is miscible with alcohol and paraffin wax: xylene
  3. Infiltrating
    - infiltrate tissues with molten paraffin wax (replacing the xylene)
  4. Embedding
    - tissues are oriented in metal moulds containing fresh molten paraffin wax and then allowed to cool
    - cooling to 4°C -> wax blocks are easily removed from metal mould, ready for microtomy/storage
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35
Q

How does an enclosed tissue processor work?

A
  1. Tissues loaded into a chamber
  2. Processing reagents are sequentially pumped in and out under vacuum to increase processing efficiency
    - > high throughput processor

=> Tissues infiltrated with paraffin wax

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36
Q

How does a paraffin wax embedding station work?

A

Specimens are transferred to a molten wax tray on the embedding station

  • tissues placed in metal moulds filled with molten wax
  • oriented optimally before placing on cold plate to set the wax
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37
Q

What is the sectioning process?

A

Process of cutting thin slices from the sample, using a microtome
- required for microscope examination

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38
Q

What is a benchtop rotary microtome?

A

Machine used to cut sections from paraffin wax embedded tissues
- thickness between 3-10 microns

39
Q

How does a benchtop rotary microtome work?

A

> Sections are floated onto water in a bath (40°C)

  • softens the wax surrounding tissue sections
  • > tissue sections become flat

> Sections may then be separated to be mounted on slides individually or as ribbons

40
Q

How does a vibrating microtome (vibratome) work?

A

> Uses embedding soft media: agarose or gelatine
Section thickness: 50-500 microns

> Sections are collected and stained as free-floating and then mounted onto slides for microscopic examination

41
Q

What are the characteristics of a cryostat?

A

> Used for sectioning frozen tissues
Sections cut with refrigerated cabinet (-20°C)
Tissue thickness: 8-50 microns

> Cut sections can immediately fixed (if frozen as fresh tissue) or stored
-20°C for short term storage ; -80°C for long term

> Vitreous water is hard enough for cutting sections
Embedding media may be used if required

> Useful for delicate or small samples
- uses an optimal cutting compound (OCT)

42
Q

What is an optimal cutting compound (OCT)?

A

Specialised embedding media that freezes at the same density as most soft tissues

43
Q

What are the characteristics of a sliding microtome?

A

> Used to section frozen samples without the need for a relatively more expensive cryostat

> Fitted to the benchtop

> Tissue is kept frozen by blasting with Co2 gaz or solid Co2 (dry ice)

> Can produce 15-200 micron sections which are stained as free-floating sections

44
Q

What is the technique of dye staining?

A

> One of the oldest histological technique available
- different formulations -> different dyes have strong affinities to particular tissue/cellular contents

> They are selective

  • e.g. crystal violet can be used to visualise presence of Nissl substance in neurons
  • > stain will react to all neurons in the brain
  • > best for studying cellular patterns in a brain area but not detailed morphology of specific neurons
45
Q

How is colour or contrast induced in tissue sections for microscopic examination?

A

Techniques that use dyes, heavy metals, and fluorochrome

46
Q

What are aniline dyes?

A

Synthetic dyes originally made from the aniline obtained from cold tar

47
Q

What is Nissl staining?

A

Dye staining technique achieved mainly by ionic interaction between dye and the tissue component

48
Q

Who developed the Nissl staining technique?

A

German pathologist Franz Nissl at the end of 19th century

- still used to identify neurons

49
Q

How does the Nissl technique work?

A

> Proteins are produced in the rough endoplasmic reticulum (RER) (within nucleus of cell)

> RER forms a large granular bodies and RNA in these granule can be visualised with basic aniline dyes (e.g. crystal violet)

> Basic / cationic dyes in aqueous solution can ionise to form net positively charged dye molecules (cations) that will combine with anions in the cell, particularly RNA and DNA

> DNA and RNA actively synthesise proteins
-> aniline dyes and Nissl bodies will stain more intensely the cationic components that have lower densities of charge

50
Q

What are the applications of Nissl staining?

A

> Study neuronal loss
- e.g. loss of CA1 neurons in hippocampus, following ischemic damage

> Study of abnormal growth and development of cerebral cortex
- e.g. mouse model of lissencephaly

51
Q

What is lissencephaly?

A

Neurological disease caused by defective neuronal migration, resulting in lack of development of the brain grooves, folds, or sulci and gyri

52
Q

What is luxol fast blue?

A

Dye solution used to visualise CNS myelin sheaths in paraffin wax sections

53
Q

How does the luxol fast blue staining work?

A

Ionic interaction between dye (anion) and myelin lipoproteins (cation) initiates binding
-> myelin sheath in blue

54
Q

What are the applications of the luxol fast blue staining?

A

> Study of the myelinated nerve tracts in CNS: structure and morphology
- e.g. combination of luxol fast blue and crysel violet staining techniques allows visualisation of both neuron cell bodies and their myelinated axons -> entire brain structure

> Study the changes that may occur to myelination
- e.g. demyelination (loss of myelin) due to injury

55
Q

What is the Golgi stain?

A

Metal impregnation technique developed by Camillio Golgi

- first published in 1883 and then modified by Santiago Ramon y Cajal

56
Q

How does the Golgi stain technique work?

A

> Stains 1 to 10% of neurons in dark brown or black, with little background staining

  • > high contrast of fine structures (e.g. dendritic spines)
  • colour is produced by silver deposits in neuron
  • only some neurons react

> Silver precipitate produces insoluble black reaction product
Small piece of formalin fixed tissue is immersed in potassium chromate, then silver nitrate

57
Q

What are the applications of the Golgi stain?

A

> Study neuronal morphology
- e.g. reconstruct the neuron and see the effects of neurodegenerative disease on neuronal morphology

> Can be used on very thick sections of the brain, or even whole brains
-> for some neuron types, you can visualise the entire neuron and most of their processes

> Used to quantify the number of dendritic spines a neuron has
- e.g. loss of prefrontal pyramidal neurons in patients with schizophrenia

58
Q

What are immunotechniques and how do they work?

A

> Techniques that visualise specific molecular targets, mostly proteins in tissues = antigens

> Antigens are visualised by use of antibodies raised or made against the antigens

> If an antigen (protein) is present in a tissue section:
- anti-protein antibodies applied to this section will bind to the antigen
- and generate an anti-proteinX / protein X complex
(antibody-antigen complex)

59
Q

What is immunohistochemistry?

A

Enzyme based detection method:
- process where an enzymatic reaction is used to visualise antibody-antigen complexes (anti-proteinX / protein X complexes) on cells grown in a laboratory, in a tissue culture

60
Q

What is immunofluorescence?

A

Fluorescence based detection method:
- visualise the antibody-antigen complexes (anti-proteinX / protein X)

= immunolabeling of cultured cells by fluorescent dyes

61
Q

On which interaction do imumunodetection methods take advantage of? What does it allow?

A

> Interaction between antibodies and their molecular targets in tissues: antigens

> Makes it possible to distinguish between cell types AND visualise the location of proteins
(e.g. cytoplasm, cell surface)

62
Q

What is an antigen?

A

Molecule which induces an immune response in our bodies
- in particular the production of antibodies

  • has protein component: glycoprotein or lipoprotein
63
Q

What is an epitope?

A

Small sequence or part of the antigen recognised by the antibody
- 8-15 amino acid sequence

-> Each antigen has many potential epitope sites

64
Q

What are monoclonal antibodies?

A

Single antibody type that specifically recognises a single epitope on antigen molecule

65
Q

How are monoclonal antibodies produced?

A

By immunising an animal -> injection of antigen in animal

  1. B cells (antibody-forming cells) are extracted from the animal’s spleen
  2. Fusion of B cells with myeloma cells (immortal cells that can be grown indefinitely in vitro
    - > generates hybridoma cells: some will generate the antibodies that interact with the specific epitope on our protein (antigen) of interest, used for immunisation
  3. Isolation and cloning of individual hybridoma cells to generate a population of desired hybridoma cells

=> monoclonal antibodies

66
Q

What are B cells, myeloma cells and hybridoma cells?

A

> B cells = antibody-forming cells

> Myeloma cells = immortal cells, can be grown indefinitely in vitro

> Hybridoma cells = can also be grown indefinitely in vitro AND can secrete antibodies

67
Q

What are memory B cells?

A

Cells that recognise the same antigen

-> enabling faster antibody production if the same antigen is seen again

68
Q

What are polyclonal antibodies?

A

Antibodies that recognise different epitopes of the same antigen molecule

69
Q

How are polyclonal antibodies produced?

A
  1. Injection of antigen in animal (usually rabbit)
  2. Animal’s B cells produce antibodies against antigen
    - polyclonal antibodies are in the animal’s serum
70
Q

Why may polyclonal antibodies cross react with multiple proteins (antigens)?

A

The more epitopes are recognised by an antibody mixture, the more opportunity that any one particular epitope - amino acid sequence - may be found on an entirely different protein

71
Q

To which protein family to antibodies belong to?

What does that say about their structure?

A

Antibodies are immunoglobulin proteins

  • > composed of 4 polypeptide chains:
  • 2 light chain copies
  • 2 heavy chain copies
72
Q

What do “light” or “heavy” polypeptide chains refer to?

A

The molecular weight of a polypeptide chain

73
Q

What are the 5 different types of antibodies?

A

> Antibodies are immunoglobulins

  • IgG
  • IgM
  • IgA
  • IgE

> most antibody reagents are IgG or occasionally IgM

74
Q

What is common to the direct and indirect methods of immunohistochemistry?

A

Antibodies are applied to the tissue section

75
Q

What is the direct method of immunohistochemistry?

A

> Primary antibody binds directly to antigen
- direct link between antibody to reporter molecule
(allows us to visualise the bound antibody)

> Only suitable for highly expressed proteins
- if there are few epitopes available, reporter signal may be too week for us to see

76
Q

What is a reporter molecule in immunohistochemistry?

A

An enzyme or a fluorochrome

77
Q

What is the indirect method of immunohistochemistry?

A

> Secondary antibodies are linked to the reporter molecule and bind specifically to primary antibody

> Secondary antibodies are polyclonal
-> will react with epitopes all over primary antibody

> As all secondary antibodies have a reporter molecule
-> signal amplified

78
Q

What is a fluorochrome?

A

Reporter molecule that will emit coloured light at a specific wavelength in visible spectrum, when using UV light
- fluorescence microscopes allow us to view the fluorochromes

79
Q

What constitutes the fluorescence detection methods?

A

> Primary or secondary antibody is linked to fluorochrome

> Using 2 or more antibodies each linked to different fluorochromes - emit light at different wavelength

> Used to detect the presence of more than one protein at the same time

80
Q

What constitutes enzyme-based detection methods?

A

> Primary or secondary antibody linked to enzyme
- most often to horseradish peroxidase (HRP)

> Substrate (chromogen) is added to the tissue

> Enzyme and substrate interact to generate an insoluble coloured product, at the site of antigen-antibody complex
- visualised under light microscope

81
Q

What are the 3 phases in immunostaining of tissue sections?

A
  1. Incorporation of positive and negative controls
  2. Antigen retrieval
  3. Blocking of non-specific binding
82
Q

Why are positive controls incorporated in tissue sections?

A

To assess fidelity of technique and specificity of primary antibody

e. g. BrdU staining in hippocampus to assess neural stem cell proliferation (stem cell divisions)
- we can confirm that our anti-BrdU antibody and staining protocol worked by including a positive control
- > presence of staining means BrdU was incorporated and staining procedure worked even if not seen in treated animal hippocampus

83
Q

What is bromodeoxyuridine (BrdU)?

A

> Synthetic analogue of the nucleotide thymidine (one of the 4 bases of human DNA)

> Used to identify dividing cells in hippocampus

> When injected in animal’s brain, it can be taken up by cells and incorporated into their DNA in place of thymidine when cells divide

84
Q

Why are negative controls incorporated in tissue sections?

A

> It is mostly sufficient to omit primary antibody (using normal serum from animal that secondary antibody was raised in instead
- any positivity seen is then assumed to be caused by non-specific binding of visualisation reagents

=> One compares the results of negative controls and positive controls against the test result before drawing a conclusion

85
Q

How can fixation procedures alter epitopes?

A

Can mask or alter epitopes so that they can no longer bind to the primary antibody

86
Q

What is an antigen unmasking/retrieval?

A

Any technique where the masking of an epitope is reversed so that the antibody can again bind to it

87
Q

What are the 2 antigen unmasking/retrieval methods?

A
  1. Heat induced epitope retrieval (HIER)

2. Protease-induced epitope retrieval

88
Q

What is the protocol for a heat induced epitope retrieval (HIER)?

A

> Buffer solutions are used to carry out HIER
- they resist to pH changes

> HIER can be performed using microwave ovens, pressure cookers, steamer, water baths
- it’s recommended to use commercial systems specifically designed for HIER

89
Q

What is the protocol for a protease-induced epitope retrieval?

A

> Sections are pre-incubated in enzyme

  • proteinase K
  • Trypsin
  • Pepsin

> Choice of enzyme and time used are determined by trial and error
- over-digestion destroys the antigen and tissue section

> This method is shown to only work for small proportion of antigens

90
Q

Why do we block non-specific binding when immunostaining tissue sections?

A

Antibodies we use can sometimes bind to non-specific components in cells and tissues with low affinity (strength)
-> false-positive signal, more often with polyclonal antibodies

=> Preventing false-positive signals

91
Q

How do we block non-specific binding in the immunostaining of tissue sections?

A

Excess proteins are added that will compete and block binding to the non-specific components in cells and tissues
- serum or excess protein-BSA (bovine serum albumin)

92
Q

What is the serum used to block non-specific binding in the immunostaining of tissue sections?

A

> Researchers use the serum of animal species that was used to raise secondary antibody

> Contains proteins that will bind to non-specific sites

93
Q

How does the protein-BSA (bovine serum albumin) block non-specific binding in immunostaining work?

A

Competes with antibodies for non-specific binding sites

- very effective

Techniques in Neuroscience (6 decks)

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