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Cell and tissue labelling 1:Antibodies and fluorophoresChoices, choices, choices
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Outline
• How labelling works• choices of antibodies• Choices of fluorophores• Limitations of standard techniques
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Antibodies 1: Choice of Primary antibody• A specific! high affinity! well characterized antibody• Generally for qualitative immuno fluorescence the following applies:
Affinity purified Polyclonal IgG to entire native protein
Affinity purified Polyclonal IgG to large fusion protein
Monoclonal IgG purified from Ascites to native antigen
Affinity Purified Polyclonal IgG to Unique Peptide
Sequence
Purified Monoclonal IgG to Unique Peptide Sequence
Non-Purified Polyclonal Serum to any antigen
BEST
WORST
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Smileytein Polyclonal antibody to native protein generates numerous markers to different protein domains
Affinity purification removes non-specific antibodies either by homologous or heterologous purification
X
X
This production method may contain antibodies which are specifically
directed to conformationally
sensitive regions of proteins. Furthermore
the entire protein acts as a potential source of
antigen sites
Affinity purified polyclonal antibodies to entire native protein
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Affinity purified polyclonal antibodies to large fusion protein
Native Smileytein Fusion protein Fusion protein may not show the same conformational structure as native protein
Affinity purification against fusion protein will generate all these antibodies
Antibodies may not bind to native protein but will bind to another unrelated molecule
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Monoclonal antibody to native antigen
Only a single antibody generated to any one antigen
With affinity purified polyclonals this may be many many different antibodies
•Monoclonals may not work if fixation effects antigen structure, •Monoclonals may be much less sensitive than polyclonal antibodies•Monoclonals may not work if antigen is lost due to complex formation
X
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Polyclonal antibody to unique peptide antigen
Generally only a short peptide (20-30 amino acids)Appropriate protein folding is rare
In our hands peptide antibodies are rarely specific even after affinity purification
Often work well on Western blot, where protein is linearised but not in immunohistochemistry
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Antibodies 2: Choice of Primary antibody
• For Quantitative Labelling – generally one epitope, one molecule therefore use a
monoclonalMonoclonal IgG purified from Ascites to native antigen
Monoclonal Culture Supernate to native antigen
Purified Monoclonal IgG to Unique Peptide Sequence
BEST
WORSTHOWEVER!!! Need to consider detection system.For example, indirect labelling (using secondary) uses a polyclonal fluorescent conjugate to detect the primary antibodyTherefore generally use directly conjugated primary monoclonal antibodies!
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Antibodies 3: Choice of Secondary antibody
• Direct immuno-conjugate of primary antibody
– Single step label
– Allows detection of protein in same species as primary antibody• eg: can detect a mouse antigen with a mouse monoclonal
• Allows double labelling with two primary antibodies from the same species
• Most quantitative
– Least amplification
– Least sensitive
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Antibodies 4: Choice of Secondary antibody
• Specific secondary immunoconjugate– Most commonly used method– Allows a single secondary to be used for multiple
primary antibodies– Some amplification
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Antibodies 5: Choice of Secondary antibody
• Protein-chrome– Small probe High mobility Immunoconjugate– Affinity varies dependant on species source of primary
antibody
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Antibodies 6: Choice of Secondary antibody
• Streptavidin Fluorophore Tertiary label– High degree of Amplification
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ABC streptavidinbiotin conjugatedperoxidase
biotinylatedtyramide
primary antibody
biotinylated secondary antibody
streptavidin fluorochrome
Antibodies 8: Pushing the limit• recent technologies use a 5 step amplification! • NB background will be amplified with foreground!
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Use of Fluorochromes: 3Double labelling
Fluorochromes are separated by colorTherefore Emission Spectra MUST NOT OVERLAP!!!!Generally neither exitation or emission overlap
Antigen Beg: mousemonoclonal
speciesspecific secondarys
Antigen Aeg: rabbit polyclonal
Fluorescein Rhodamine
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Emission Spectra must not cross-over
Wavelength
Inten
sity
Cy 3.18 Rhodamine
Cy 3 bleeds into the Rhodamine emissionSpectra
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Routinely used fixed wavelength fluorophores
increasing
w
avele
ngth
Cy5Texas RedRhodamineCy3
Phycoerythrin
FluoresceinBodipy
DAPI
VISIBLE by EYE
NEED CCD to detect
Antigen Aeg: rabbit polyclonal
Fluoresceinconjugatedsecondary
AMCA
ALEXA 488
ALEXA 568
ALEXA 543
ALEXA 380
ALEXA 647
Excitation of ALEXAs not that tight. Match Excitation with laser
wavelength
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Typical triple color:
Red = ActinBlue = NucleiGreen = Peroxisomes
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Cell and Tissue labelling:2 Practical aspects
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Fixation
• Needs to be:– Fast– Preserve structure– Not fluorescent– Preserve antigenicity
• Two main types– Cross linkers– Dehydrators
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• Glutaraldehyde – EM fix, autofluorescent, changes protein shape, slow
• Formaldehyde – Simplest mono-aldehyde
– Immobilizes proteins
– Fast
– Exists as a glycol in solution
– Use as 2% solution
– Does not permeabilize cells, no good for lipids
– 10 minutes for cells, one hour for tissues, fix by perfusion if possible
CH3-CO
H+ RNH2 CH3-C
NR
H+ H2O
Cross linkers:
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Paraformaldehyde recipe
• 8 gms paraformaldehyde resin• 70 mls water• Heat to 70 Celsius (no more)• Add base until it goes clear (2 or 3
drops)• Cool• Add 30 mls 0.3M PBS• pH• Filter• Store at 4 Celsius • Dilute to 2% in PBS prior to use
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Sticking sections onto slides
• Superfrost slides• 0.2% Gelatin (300 bloom) in water• Poly-L-lysine solution• Cell-Tak• Alcian Blue
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Dehydrators• COLD methanol -20oC• COLD acetone
– remove lipid– often change protein shape– soluble proteins may stick to cytoskeleton
giving erroneous distribution– great for nuclear label– only useful on cells or on sections after they are
cut– use paraformaldehyde w/ 0.1% triton preferably
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Immunolabelling 1: Blocking• First blocking steps for free protein binding sites
such as unsaturated aldehydes and other “unknowns”
• We use a mix of 0.1M PBS
5.0% Bovine Serum Albumin 0.1% Glycine • “Blocking Buffer” • 3 X 5 minutes• Block non-specific antibody binding sites:
– 5% normal goat serum in Blocking Buffer for 30 minutes
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Immunolabelling: primary antibody
• Following Blocking antibody step go straight into primary antibody– Generally use at about 1-2 g/ml
– dilute antibody in Blocking Buffer
– Spin antibody in Microfuge (13,000g X 10 minutes) before use
– incubate 1 hour (occasionally overnight)
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Immunolabelling 5:secondary antibody
• Following primary antibody wash well in Blocking buffer– At least! 3 X 5 minute washes
• Secondary antibody– dilute in blocking buffer
• Varies according to manufacturer• need to do dilution series first
– spin to remove aggregates • 4 minutes 13,000G nm
– Need about 50l/slide – Incubate 30 minutes- 1hour
• Following Secondary antibody– 3 washes in blocking buffer– 3 washes in PBS
• Mount: use aqueous mount, we use Gelvatol
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Gelvatol• Materials:• PVA- Sigma Chemical Cat. #P-8136• Glycerol- Sigma Chemical Cat. #G-9012• Sodium Azide- Fisher Chemical Cat.
#S227-100– Add 21 g PVA to 42 mL glycerol.– Add 52 mL dH20.– Add a few crystals of sodium azide.– Add 106 mL Tris (.2M, pH=8.5).– Stir with low heat for a few hours or until
reagents dissolved.– Clarify the mixture by centrifugation at
5000g for 15 minutes.– Aliquot and store either at 4 degree C.
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Viscosity
• The viscosity should be like honey (commercial products like Molviol are too thin)
• Add more PVA in step 4 until the solution becomes the desired viscosity. Suggest putting the beaker of gelvatol in the refrigerator overnight, after step 5 and before step 6, and check it in the morning to be sure that the viscosity is correct. If it is, continue on to step 6. If it too viscous, add a little more glycerol to bring the viscosity down and then go on to step 6. If it is not viscous enough, add more PVA with heat and refrigerate for a few more hours to check the viscosity before going on to step 6.
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Controls
• Pre-immune serum• No primary antibody• Irrelevant primary antibody• Dilution series
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Artifacts
• Two causes– Fixation
• Aldehydes are autofluorescent, remove with NaBh4 wash (10 mins, 0.1%, kind of aggressive though!)
– cellular components• This is caused by fat deposits such as
lipofucsin, enzyme granules, lysosomes etc etc. • This can be selected and removed using image
processing and visually using a dual pass cube
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Autofluorescence
Red cube Green cube
Dual pass cube
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Artifacts 2: bleed throughA Tight red
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Bleed through, green into red
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0secs 30 secs
60 secs
Bleaching of fluorophores (Phycoerythrin)
Use fluorophores that resist bleaching (Alexa’s, carbocyanines)Gate exposures.
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Registration errors
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TUNEL and DNA stain(Hoescht dye) within cultured cells
Using dual pass, blue green cubeSuperposition of independentgreen and blue images
Problem is caused by fractional differences in the angle of the dichroic
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