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The Aaron Klug Centre for Imaging and Analysis

Lecture 4: Intracellular localisation of biological molecules

There are a number of websites and files that contain supplementary information to my lecture that you should visit:

  1. Enzyme cytochemistry.
  2. Probes for identifying actin
  3. Probes for identifying other cellular compartments. Pay particular attention to lectins and their uses.
  4. Use of tyramide for amplifying immunolabelling and cytochemical signals.
  5. The uses of chicken immunoglobulins (IgY). Browse this website to find out more information on antibody production, purification, modification and assay development. Of all the websites, this is the most important as it provides a good introduction to immunochemistry.
  6. Gold colloids and various derivatives available. Also, check out the page on silver enhancement of colloidal gold.

Some of the questions you should consider while going through this section are:

  • What methods are available for detecting the biological activity of molecules? What problems are associated with these methods?
  • What compounds can one attach to antibodies? What is the chemistry behind fluorophore attachment versus gold colloids?
  • How does one go about choosing the correct fluorophore wavelength when embarking on an immunofluorescence experiment?
  • What controls are necessary in a localisation experiment?
  • What are the benefits and pitfalls of autoradiography?
  • How would use ensure that a cytochemical reaction (e.g., assaying a phosphatase within a cell) is specific?
  • Why do reaction products from cytochemical reaction have to be insoluble?
  • How would you optimise the amount of fixative to use on cells that were going to be processed for enzyme cytochemistry?
  • What information does an ELISA provide? What does antibody "titre" refer to?
  • Why is the enzyme product in an ELISA soluble, but insoluble in western blots and enzyme cytochemistry?
  • Why does one immunise experimental animals more than once when raising antibodies? What would happen if they were only immunised once?
  • Why are adjuvants used?
  • Why would you want to purify IgG or IgY from total serum or egg yolk?
  • Can you use fluorescent-labelled antibodies on cells that are going to be viewed under the TEM? Why? Why not?
  • Can you use gold-labelled antibodies on cells that are going to be viewed under the light microscope? Why? Why not?
  • Why do you need to perform western blotting before immunocytochemistry?
  • Would you use reducing or non-reducing SDS-PAGE for western blotting? Why?
  • What are the advantages of monoclonal antibodies over polyclonal antibodies and vice versa?
  • How do fixatives work - what are the differences between aldehyde fixatives and osmium tetroxide, alcohols and potassium permanganate?
  • What is osmolarity? Why is it important when designing a fixation protocol?
  • When would you want to cryofix and freeze substitute samples as opposed to regular wet fixation and resin embedding?
  • How are colloidal gold probes produced? Is the size of the gold colloid important when designing an immunolabelling protocol?
  • How does silver enhancement work? Could you use it in light microscopy?

Intracellular localisation of biological molecules

Brendon Price, Ph.D. Electron Microscope Unit

Structure of the lecture

  • Molecules/chemical entities that can be detected intracellularly
  • Microscopic methods available
  • Localisation by exploiting biological activity
  • Localisation using immunochemistry
  • Caveats
  • Interpretation of micrographs

Molecules/chemical entities that can be detected intracellularly

  • Chemical
    • simple, e.g. [Ca2+]i, vitamin
    • complex, e.g. drug
  • Biological polymer
    • protein
    • lipid
    • region of DNA, mRNA transport
  • Intracellular compartment
    • Golgi complex, endosome, mitochondrion

Microscopy methods available

  • Light microscopy
    • Brightfield
    • Darkfield
    • Fluorescence
  • Electron microscopy
    • SEM, incl. EBSD
    • TEM, incl. HVTEM, EELS

Which method to use?

  • What resolution/detail/information is required?
  • Examples:
    • locate & track nuclear ultrastructural changes during cell death/apoptosis
    • locate & quantify mitochondria
    • determine whether a lipid exists on the inner or outer leaflet of an organelle membrane

Localisation by exploitation of biological activity

How do we accurately identify a particular compound/molecule amongst thousands of other biochemicals within a single cell?

Option #1: We could exploit the biological activity of the target

  • Enzyme reactions
    • add substrate
    • product rendered visible and insoluble
  • Binding of unique co-factor/substrate
    • co-factor/substrate is labelled and added to cells
    • remain "locked" during fixation
    • require v. low Kd

Other unique properties to exploit

  • pH , e.g. acidic organelles with ionisable probes
  • membrane potential of mitochondria between inner and outer membranes
  • lipid fluidity of organelles, e.g., BODIPY-ceramide labelling of Golgi

Conditions that must be met:

  • the product or substrate or co-factor must not be destroyed or occluded by fixation
  • the protein/lipid structure must not be compromised by fixation
  • the labelled product must be stable and visible

Localisation using immunochemistry

Option #2: We could exploit the specificity of antibodies

  • This is particularly useful when:
    • biological activity of the target is not preserved
    • activity assay is not suitable for LM or EM
    • biological activity is not known; intracellular localisation may predict function

Antibodies 101

  • Bivalent molecules responsible for classical pathway of complement activation
  • Highly selective, stable
  • 2 heavy chains, 2 light chains
  • Monoclonal or polyclonal
    • depends on epitope target, budget & application

Antibodies can be coupled to compounds that are visible on both light and electron microscopes

Antibodies can be raised against tags:

  • 6xhistidine
  • FLAG
  • biotin
  • SAVES A LOT OF TIME AND MONEY
  • but - you must ensure that tag doesn't affect structure or sorting e.g, C-term Val

The long walk to immunocytochemistry success

  • Purification of antigen
  • Inoculation into experimental animals
  • Assessment of antibody response
  • Antibody purification
  • Assessment of antibody specificity
  • Assessment of antigen sensitivity to fixation
  • Choice of resin

Purification of antigen

  • Non-protein molecules should be purified to homogeneity as assessed by MALDI, TLC or HPLC
  • Proteins should be purified to homogeneity as assessed by reducing SDS-PAGE and silver staining
  • Products should be sterile and endotoxin-free

Small molecules and proteins <10 kDa will not usually elicit an immune response and require coupling to a carrier protein

  • KLH (keyhole limpet haemocyanin)
  • BSA
  • Coupling via N- or C-terminus, sulfhydryl groups

How much protein do I need for the entire procedure?

  • Ideally 1 mg pure protein
  • But, immunogenicity is protein-specific
  • Do not inoculate too much protein or you may induce tolerance

Preparation for inoculation

  • Protein must be stably emulsified in an adjuvant
  • Adjuvant = mineral oil
  • Adjuvants create a "depot" effect and allow the protein to persist in the body
  • Continual stimulation of immune system
  • 2 types of adjuvant
    • Freund's incomplete - mineral oil
    • Freund's complete - mineral oil + heat killed M. tuberculosis
    • Complete adjuvant is ONLY used in the first inoculation
    • Incomplete is used for the booster injections
    • Other adjuvants - aluminium hydroxide

Choice of animal

  • How much antibody do you need?
  • Are you concerned about cross-species reactivity?

Inoculation procedure

  • Rabbit
    • subcutaneous
    • test bleeds from ear vein
    • final blood collection usually by exsanguination
    • IgG is present in the serum
  • Chicken
    • intramuscular
    • IgY is packaged into the yolks of eggs
    • no test bleed required
    • much more antibody than rabbits

So, why use rabbits?

  • Not many people know about chickens
  • Most EM immunocytochemistry takes advantage of protein A gold
    • Staphylococcus aureus protein A shows a high affinity for rabbit IgG, particularly the heavy chain Fc domains
    • but recently, protein L and protein G
    • No matter which animal you use, make sure that you have pre-immune antibodies before you inoculate

Assessment of antibody response

  • 1st inoculation will produce IgM
  • Require further injections to induce class shift and increase titre
  • Usually 1 primary & 3 boosters over 12 week period
  • Response is assessed by indirect ELISA

Antibody purification

  • Ion exchange
    • unusually high pI of IgG
  • Dextran sulfate
  • Polyethylene glycol 6000 precipitation
  • After purification, estimate conc. by A280 using extinction coefficient =1.64

Assessment of antibody specificity

  • Western blotting
    • prepare homogenates - soluble? membrane-associated?
    • Boil samples in the presence of reducing agents
    • Separate by SDS-PAGE under reducing conditions
    • Transfer to solid support
    • Probe with labelled secondary antibody and detect with enzyme label

Assessment of antigen sensitivity to fixation

  • Fixatives can perturb the tertiary structure of some proteins
  • May destroy conformationally-dependent epitopes, esp. with monoclonals. Contiguous OK
  • Choose PFA 1-8%, glut 0.05-2% in a buffer at correct pH
  • Fix transferred proteins & repeat w.blot to ensure no destruction

Choice of resin

  • Do not use plastic resins
  • Do not osmicate your tissue
  • Methacrylate resins the best
    • LR White
    • Lowicryl
  • Low temp embedding resins
    • Lowicryl, LR Gold

Caveats and labelling interpretation

  • ICC can be highly subjective. Do not be a slave to your own bias or your supervisor's
  • No labelling can be a good thing
  • Get to grips with stereology
  • Beware of artefacts

Miscellaneous

  • Marker proteins
    • dep. on cell-type
    • reference point for localisation
    • e.g., lactoferrin in neutrophils
    • von Willebrand factor in endothelial cells
  • Tyramide amplification of signal
    • HRPO-mediated free radical attack
    • tyramine can be conjugated to BSA-gold or fluorophores

Antigen retrieval

  • microwave
  • trypsin digestion
  • sodium metaperiodate/hydrogen peroxide

Finally...

Before even thinking of fixing your sample for ICC:

  • make sure antigen was pure
  • make sure you've purified antibody prep
  • test specificity by W.blot
  • test sensitivity to fixation by W.blot or modified ELISA
  • compare results to the pre-immune antibody prep