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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