Cell and tissue labelling 1: Antibodies and fluorophores Choices, choices, choices

1. Cell and tissue labelling 1:Antibodies and fluorophoresChoices, choices, choices

2. Outline How labelling works choices of antibodies Choices of fluorophores Limitations of standard techniques

3. Antibodies 1: Choice of Primary antibody 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 A specific! high affinity! well characterized antibody Generally for qualitative immuno fluorescence the following applies:

4. Affinity purified polyclonal antibodies to entire native protein Smileytein Polyclonal antibody to native protein generates numerous markers to different protein domains 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 X X Affinity purification removes non-specific antibodies either by homologous or heterologous purification

5. Affinity purified polyclonal antibodies to large fusion protein Native Smileytein Fusion protein Antibodies may not bind to native protein but will bind to another unrelated molecule Fusion protein may not show the same conformational structure as native protein Affinity purification against fusion protein will generate all these antibodies

6. 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

7. 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

8. Antibodies 2: Choice of Primary antibody Monoclonal IgG purified from Ascites to native antigen Monoclonal Culture Supernate to native antigen Purified Monoclonal IgG to Unique Peptide Sequence BEST WORST HOWEVER!!! Need to consider detection system. For example, indirect labelling (using secondary) uses a polyclonal fluorescent conjugate to detect the primary antibody Therefore generally use directly conjugated primary monoclonal antibodies! • For Quantitative Labelling • generally one epitope, one molecule therefore use a monoclonal

9. 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

10. 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

11. Antibodies 5: Choice of Secondary antibody • Protein-chrome • Small probe High mobility Immunoconjugate • Affinity varies dependant on species source of primary antibody

12. Antibodies 6: Choice of Secondary antibody • Streptavidin Fluorophore Tertiary label • High degree of Amplification

13. Antibodies 8: Pushing the limit streptavidin fluorochrome biotinylated tyramide biotinylated secondary antibody primary antibody ABC streptavidin biotin conjugated peroxidase recent technologies use a 5 step amplification! NB background will be amplified with foreground!

14. Fluorescein Antigen A eg: rabbit polyclonal Use of Fluorochromes: 3Double labelling • Fluorochromes are separated by color • Therefore Emission Spectra MUST NOT OVERLAP!!!! • Generally neither exitation or emission overlap species specific secondarys Rhodamine Antigen B eg: mouse monoclonal

15. Emission Spectra must not cross-over Rhodamine Cy 3.18 Intensity Cy 3 bleeds into the Rhodamine emission Spectra Wavelength

16. Fluorescein conjugated secondary Antigen A eg: rabbit polyclonal Routinely used fixed wavelength fluorophores NEED CCD to detect Cy5 Texas Red ALEXA 647 Rhodamine Cy3 ALEXA 568 Phycoerythrin ALEXA 543 increasing wavelength Fluorescein VISIBLE by EYE Bodipy ALEXA 488 DAPI ALEXA 380 AMCA Excitation of ALEXAs not that tight. Match Excitation with laser wavelength

17. Typical triple color: Red = Actin Blue = Nuclei Green = Peroxisomes

18. Cell and Tissue labelling:2 Practical aspects

19. Fixation • Needs to be: • Fast • Preserve structure • Not fluorescent • Preserve antigenicity • Two main types • Cross linkers • Dehydrators

20. O NR CH3-C + RNH2 CH3-C + H2O H H Cross linkers: • 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

21. 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

22. Sticking sections onto slides Superfrost slides 0.2% Gelatin (300 bloom) in water Poly-L-lysine solution Cell-Tak Alcian Blue

23. 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

24. 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

25. Immunolabelling: primary antibody • Following Blocking antibody step go straight into primary antibody • Generally use at about 1-2 mg/ml • dilute antibody in Blocking Buffer • Spin antibody in Microfuge (13,000g X 10 minutes) before use • incubate 1 hour (occasionally overnight)

26. 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 50ml/slide • Incubate 30 minutes- 1hour • Following Secondary antibody • 3 washes in blocking buffer • 3 washes in PBS • Mount: use aqueous mount, we use Gelvatol

27. 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.

28. 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.

29. Controls Pre-immune serum No primary antibody Irrelevant primary antibody Dilution series

30. 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

31. Autofluorescence Red cube Green cube Dual pass cube

32. Artifacts 2: bleed through A Tight red

33. Bleed through, green into red

34. CBI Bleaching of fluorophores (Phycoerythrin) 30 secs 0secs Use fluorophores that resist bleaching (Alexa’s, carbocyanines) Gate exposures. 60 secs

35. CBI Registration errors TUNEL and DNA stain(Hoescht dye) within cultured cells Superposition of independent green and blue images Using dual pass, blue green cube Problem is caused by fractional differences in the angle of the dichroic