Introduction to immunohistochemistry Flashcards
What do histological techniques allow us to study?
> Tissue anatomy and cytoarchitecture
Distribution of proteins
Pathological changes associated with disease
In which fields is the histological study of tissues essential in?
> Clinical diagnostic neuropathology
> Basic and translational neuroscience research
What are the two sources of tissue for histological studies?
- Animal models
2. Human: post-mortem donor tissue, pathology samples, surgical surplus
What are the advantages of animal models?
> Can study different stages of disease
Can study effects of specific mutations
Can access therapeutic strategies
What are the limitations of animal models?
> May not fully recapitulate human disease
> Ethical concerns
What are the advantages of human tissues for histological studies?
> Reduce need for animal research
> Arguably better for studying human disease
What are the limitations of human tissues for histological studies?
> Ethical concerns
Limited tissue supply
Low availability of early stages of disease
What happens to the tissue when removed from living organism or deceased donor?
Irreversible processes of autolysis (self-destruction) and necrosis begin
-> cellular damage
What is the difference between tissue from post-mortem donor and living donor?
Tissue from post-mortem donor more likely to have signs of cellular damage than samples from living donors
What are the two aims of tissue preservation for histology?
- To preserve the tissue in as life-like manner as possible
2. To prevent irreversible cell/tissue destruction
What are the common methods of tissue preservation?
> Chemical fixation
> Cryopreservation
What are the various chemical fixatives?
- Acetic acid
- Formaldehyde
- Ethanol
- Glutaraldehyde
- Methanol
- Picric acid
What does the choice of chemical fixative for preservation of tissue depend on?
The requirements from the experimental design
Why not use the chemical fixatives glutaraldehyde, acetic acid or methanol for tissue preservation?
> Glutaraldehyde -> best morphology, poor staining
> Acetic acid, methanol -> poor morphology, best staining
Why is formaldehyde the most common chemical fixative used to preserve tissue?
Offers best balance between morphology and staining quality
What are the effects of chemical fixatives?
Stabilise proteins and other macromolecules
What are the 2 categories of chemical fixatives?
- Cross linking fixatives
2. Precipitating fixatives
Which chemical fixatives are cross linking fixatives? What is their process of tissue preservation?
Formaldehyde and glutaraldehyde:
- create covalent bonds between proteins in the tissue
-> good preservation of morphology BUT slow fixation process
What are aldehydes and what are their action on cells?
Organic compounds that anchor proteins relative to each other inside the cells and between cells
Which chemical fixatives are precipitating fixatives? What is their process of tissue preservation?
Ethanol and methanol:
- disrupt hydrophobic bonds between proteins causing them to irreversibly precipitate
-> less suitable for antibody-based techniques: immunohistochemistry
What is a protein tertiary structure?
Three-dimensional structure of a protein
What are hydrophobic bonds?
They arise from the interaction of the hydrophobic amino acids with water
What are the methods of tissue fixation?
- Immersion fixation
2. Perfusion fixation
What is the method of immersion fixation?
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
What is the method of perfusion fixation?
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
What are the factors affecting the quality of tissue fixation?
> Changes in pH (acidity)
Length of incubation in fixative
Specimen size (no larger than 5mm3 for optimal fixation)
Temperature
How does temperature affect the quality of tissue fixation?
> 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
What does cryopreservation of tissue consist of?
Preservation of tissue structure and components by freezing them rapidly without fixation
- snap-freezing the sample with dry ice or liquid nitrogen
What are the advantages of cryopreservation?
> Fastest method
Minimal changes to protein structure
Rapid cooling (-70°C using liquid nitrogen) minimises damage to the tissue (= ice-crystal artefact)
How does the rapid cooling with liquid nitrogen in cryopreservation minimises tissue damages?
-70°C using liquid nitrogen:
liquid water is converted to vitreous water without going through crystalline phase
What are the disadvantages of cryopreservation?
> 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
Why are tissue samples embedded in a solid medium?
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
Why is paraffin wax the most common embedding media of tissue samples?
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
What are the 4 phases for processing fixed tissue to paraffin wax?
- Dehydrating
- sequential immersions in alcohol - Clearing
- remove alcohol in solvent that is miscible with alcohol and paraffin wax: xylene - Infiltrating
- infiltrate tissues with molten paraffin wax (replacing the xylene) - 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
How does an enclosed tissue processor work?
- Tissues loaded into a chamber
- Processing reagents are sequentially pumped in and out under vacuum to increase processing efficiency
- > high throughput processor
=> Tissues infiltrated with paraffin wax
How does a paraffin wax embedding station work?
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
What is the sectioning process?
Process of cutting thin slices from the sample, using a microtome
- required for microscope examination
What is a benchtop rotary microtome?
Machine used to cut sections from paraffin wax embedded tissues
- thickness between 3-10 microns
How does a benchtop rotary microtome work?
> 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
How does a vibrating microtome (vibratome) work?
> 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
What are the characteristics of a cryostat?
> 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)
What is an optimal cutting compound (OCT)?
Specialised embedding media that freezes at the same density as most soft tissues
What are the characteristics of a sliding microtome?
> 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
What is the technique of dye staining?
> 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
How is colour or contrast induced in tissue sections for microscopic examination?
Techniques that use dyes, heavy metals, and fluorochrome
What are aniline dyes?
Synthetic dyes originally made from the aniline obtained from cold tar
What is Nissl staining?
Dye staining technique achieved mainly by ionic interaction between dye and the tissue component
Who developed the Nissl staining technique?
German pathologist Franz Nissl at the end of 19th century
- still used to identify neurons
How does the Nissl technique work?
> 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
What are the applications of Nissl staining?
> 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
What is lissencephaly?
Neurological disease caused by defective neuronal migration, resulting in lack of development of the brain grooves, folds, or sulci and gyri
What is luxol fast blue?
Dye solution used to visualise CNS myelin sheaths in paraffin wax sections
How does the luxol fast blue staining work?
Ionic interaction between dye (anion) and myelin lipoproteins (cation) initiates binding
-> myelin sheath in blue
What are the applications of the luxol fast blue staining?
> 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
What is the Golgi stain?
Metal impregnation technique developed by Camillio Golgi
- first published in 1883 and then modified by Santiago Ramon y Cajal
How does the Golgi stain technique work?
> 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
What are the applications of the Golgi stain?
> 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
What are immunotechniques and how do they work?
> 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)
What is immunohistochemistry?
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
What is immunofluorescence?
Fluorescence based detection method:
- visualise the antibody-antigen complexes (anti-proteinX / protein X)
= immunolabeling of cultured cells by fluorescent dyes
On which interaction do imumunodetection methods take advantage of? What does it allow?
> 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)
What is an antigen?
Molecule which induces an immune response in our bodies
- in particular the production of antibodies
- has protein component: glycoprotein or lipoprotein
What is an epitope?
Small sequence or part of the antigen recognised by the antibody
- 8-15 amino acid sequence
-> Each antigen has many potential epitope sites
What are monoclonal antibodies?
Single antibody type that specifically recognises a single epitope on antigen molecule
How are monoclonal antibodies produced?
By immunising an animal -> injection of antigen in animal
- B cells (antibody-forming cells) are extracted from the animal’s spleen
- 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 - Isolation and cloning of individual hybridoma cells to generate a population of desired hybridoma cells
=> monoclonal antibodies
What are B cells, myeloma cells and hybridoma cells?
> 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
What are memory B cells?
Cells that recognise the same antigen
-> enabling faster antibody production if the same antigen is seen again
What are polyclonal antibodies?
Antibodies that recognise different epitopes of the same antigen molecule
How are polyclonal antibodies produced?
- Injection of antigen in animal (usually rabbit)
- Animal’s B cells produce antibodies against antigen
- polyclonal antibodies are in the animal’s serum
Why may polyclonal antibodies cross react with multiple proteins (antigens)?
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
To which protein family to antibodies belong to?
What does that say about their structure?
Antibodies are immunoglobulin proteins
- > composed of 4 polypeptide chains:
- 2 light chain copies
- 2 heavy chain copies
What do “light” or “heavy” polypeptide chains refer to?
The molecular weight of a polypeptide chain
What are the 5 different types of antibodies?
> Antibodies are immunoglobulins
- IgG
- IgM
- IgA
- IgE
> most antibody reagents are IgG or occasionally IgM
What is common to the direct and indirect methods of immunohistochemistry?
Antibodies are applied to the tissue section
What is the direct method of immunohistochemistry?
> 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
What is a reporter molecule in immunohistochemistry?
An enzyme or a fluorochrome
What is the indirect method of immunohistochemistry?
> 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
What is a fluorochrome?
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
What constitutes the fluorescence detection methods?
> 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
What constitutes enzyme-based detection methods?
> 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
What are the 3 phases in immunostaining of tissue sections?
- Incorporation of positive and negative controls
- Antigen retrieval
- Blocking of non-specific binding
Why are positive controls incorporated in tissue sections?
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
What is bromodeoxyuridine (BrdU)?
> 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
Why are negative controls incorporated in tissue sections?
> 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
How can fixation procedures alter epitopes?
Can mask or alter epitopes so that they can no longer bind to the primary antibody
What is an antigen unmasking/retrieval?
Any technique where the masking of an epitope is reversed so that the antibody can again bind to it
What are the 2 antigen unmasking/retrieval methods?
- Heat induced epitope retrieval (HIER)
2. Protease-induced epitope retrieval
What is the protocol for a heat induced epitope retrieval (HIER)?
> 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
What is the protocol for a protease-induced epitope retrieval?
> 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
Why do we block non-specific binding when immunostaining tissue sections?
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
How do we block non-specific binding in the immunostaining of tissue sections?
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)
What is the serum used to block non-specific binding in the immunostaining of tissue sections?
> Researchers use the serum of animal species that was used to raise secondary antibody
> Contains proteins that will bind to non-specific sites
How does the protein-BSA (bovine serum albumin) block non-specific binding in immunostaining work?
Competes with antibodies for non-specific binding sites
- very effective
