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An Introduction to Immunofluorescent Staining of Cultured Cells

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This presentation covers materials, common variations and necessary controls in a immunofluorescent staining protocol and a simple guide for troubleshooting.

This presentation covers materials, common variations and necessary controls in a immunofluorescent staining protocol and a simple guide for troubleshooting.

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  • 1. An Introduction to Immunofluorescent Staining of Cultured Cells Life Technologies Technical Support Webinar Series Presented October 5, 2011 by Jason Kilgore, Technical Applications Scientist II Life Technologies Molecular Probes Labeling and Detection Technologies Learn more – video tutorials about the basics of Fluorescence1 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 2. The Beauty of High Quality Fluorescence Images2 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 3. Topics to Be Covered In this presentation we plan to:  Discuss steps of an immunofluorescent staining protocol − material list − common variations − necessary controls  Provide a simple guide for troubleshooting − decision tree − demonstrate common pitfalls with example images3 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 4. A Standard Immunofluorescence Protocol 1. Observe 2. Fix 3. Permeabilize 4. Image-iT™ FX signal enhancer 5. Block 6. Incubate with primary antibody 7. Incubate with secondary antibody 8. Counterstain 9. Mount4 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 5. Material Required To complete this protocol, the following material is required:  A coverslip coated with healthy cells  Formaldehyde  Triton X-100  Image-iT™ FX signal enhancer (I36933)  BSA (fraction V, lipid free)  DAPI  ProLong® Gold or SlowFade® Gold5 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 6. Observe the Cells Live cells should be healthy and of an appropriate confluency  ~50–75% confluency  Evenly distributed across the coverslip  Expected morphology6 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 7. Methods of Fixation There are two types of common fixatives:  Crosslinking fixatives — Fixatives that form covalent bonds between amines. − Formaldehyde − Glutaraldehyde  Coagulating fixatives — Fixatives that precipitate proteins − Methanol − Acetone7 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 8. Secrets to Good Formaldehyde Fixation Proper handling during fixation can greatly affect the quality of staining  Always use freshly prepared high-quality formaldehyde  Use warm (37ºC) 2–4% formaldehyde, incubated with sample for 10–15 minutes  You may dilute formaldehyde in complete media for some staining applications8 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 9. Methods of Permeabilization Detergents, alcohols, and acetone disrupt membranes  Triton X-100 − 0.05–0.2% Triton X-100 at room temperature for 5 minutes  Acetone — sometimes used following the use of a crosslinking fixative − pre-chilled acetone is incubated with cells for 10 minutes at -20ºC9 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 10. Why Use Image-iT™ FX Signal Enhancer Fluorophores can interact with structures in the cell Image-iT FX™ Signal Untreated Enhancer Treated Tetramethylrhodamine staining Image-iT™ FX signal enhancer blocks this type of background.10 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 11. Blocking ReagentBlocking options 3-5% BSA (fraction V, lipid free) mixed in PBS Serum from the species in which your secondary antibody was raised Mixtures of BSA with the Cells blocked with BlockAid™, appropriate serum then stained with an anti- mitochondrial antibody and BlockAid™ blocking counterstained with green- fluorescent phalloidin and solution (B10710) DAPI11 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 12. Selection of Primary Antibody The primary antibody is the most important aspect of immunofluorescent staining  Should be tested for use in immunofluorescence  Commercial antibodies may come with protocols Anti-mitochondrial primary antibody used with phalloidin and DAPI counterstain12 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 13. Choosing a Secondary Antibody Type Pros Cons Strongest signal, Whole IgG Possible crossreactivity inexpensive Would not interact with Lower signal due to fewer F(ab)2 F(c) receptors dye molecules Highly Cross adsorbed Less crossreactive May require higher titer Expensive, must know Subtype Specific IgG1, IgG2a, IgG2b, IgG3, IgM subtype of primary Considerations:  Specificity for the primary antibody species  Avoid same-species staining  Fluorophore label must match equipment  Subtype of primary antibody13 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 14. Selection of Fluorophore The chief factor is the filter availability 1. Identify the wavelengths capable of detecting 2. Choose the corresponding fluorophore based on this information Alexa Fluor® Dye Classic Dye Ex/Em FL. Color AF350 AMCA 346/445 Blue AF488 Fluorescein, Cy2 494/520 Green AF555 Tetramethylrhodamine, Cy3 555/572 Orange AF568 Rhodamine Red 578/602 Orange-Red AF594 Texas Red® 590/617 Red AF647 Cy5 651/672 Far-Red14 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 15. The Alexa Fluor® Dyes Alexa Fluor dyes provide bright and photostable conjugates that match most filter and illumination source configurations.15 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 16. Why Use Alexa Fluor® Dyes? Photobleaching Demonstration Alexa Fluor® dyes are Fluorescein Alexa Fluor® 488 superior for Fluorescence microscopy Initial  Very bright dyes  Photostable  Less pH-sensitive exposure After than classic dyes  Matched to common filters16 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 17. Incubation with Secondary Antibody The optimal conditions must be determined empirically for every sample  Typical concentrations range from 1–10 ug/ml  Centrifuge diluted antibodies  Incubation times are usually between 30–60 minutes  Incubate in a humidified chamber17 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 18. Counterstaining Counterstaining is useful to orient observed immunofluorescence staining Common counterstains  Nucleic acid stains  Actin stains18 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 19. The Benefits of Antifade Mounting Agents Antifade reagents extend the fluorescence of a sample  ProLong® Gold — polymerizing  SlowFade® Gold — non-polymerizing With ProLong® Gold Without antifade19 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 20. Experimental Controls There are two controls that should be completed with every staining that will allow you to validate and troubleshoot the observed results 1. An autofluorescence/no-secondary antibody control 2. A no-primary antibody control Both controls should show no signal20 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 21. Troubleshooting Immunfluorescent Staining Thinking like a technical support scientist:  Decision tree  Suggested solutions Fixed Muntjac skin cells with Alexa Fluor 488 phalloidin, SYTOX Orange, and mouse anti-OxPhos Complex V Inhibitor Protein with goat anti-mouse Alexa Fluor 64721 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 22. Some Sources of Error  Autofluorescence – Look at unstained cells/tissue  Secondary Antibody Background − Too much antibody — reduce the amount of secondary − Insufficient blocking — alternate method of blocking − Dye-sample interactions — Image-iT™ FX signal enhancer − Antibody aggregates — centrifuge antibodies − Loss of specificity — replace the antibody22 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 23. Some Sources of Error  Lack of Signal − Primary antibody — test with a proven secondary − Filters — check equipment specifications − Low antigen levels — try signal amplification − Poor Fixation — try antigen retrieval − Secondary antibody — test with a proven primary antibody23 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 24. Sources of Autofluorescence Autofluorescence is fluorescence signal that comes from the sample itself  Fixation — use sodium borohydride  Endogenous cellular components − Sudan black or trypan blue − copper sulfate − photobleaching  Too strong — use different filters24 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 25. Examples of Autofluorescence Autofluorescence caused by aldehyde fixation visible through an assortment of filters25 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 26. Example of Secondary-Caused Problems Too much secondary can generate overexposed images and increased on-cell background Too much antibody Correct antibody titer26 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 27. Primary Antibody Troubleshooting If both controls show no staining, the fluorescence pattern in your samples must be due to your primary antibody  Make sure antibodies are properly stored  Centrifuge antibody prior to staining  Not all primary antibodies are suitable Correct tubulin staining with phalloidin and DAPI counterstaining27 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 28. Antigen Retrieval Some antibodies require additional steps to make the antigens available for detection- KNOW YOUR PRIMARY Staining with an antigen-retrieval–sensitive mitochondrial antibody No antigen retrieval Antigen retrieval in urea28 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 29. Conclusion  Be patient  Every staining experiment requires optimization  Run controls with every experiment  If problems still exist, please contact FluoCells® prepared slide #2 BPAE our technical cells stained with anti-tubulin primary and BODIPY® FL secondary antibodies, support scientists Texas Red®-X phalloidin and DAPI29 Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential
  • 30. Contact Technical SupportWe succeed when you succeed! U.S. Technical Support Hours: 9:00 AM - 8:00 PM EST Phone: (800) 955-6288, Option 5 FAX: (800) 352-1468 Email: techsupport@lifetech.com BPAE cells stained with Alexa Fluor 488 phalloidin and DAPIInvitrogen Canada - (800) 263 6236 Learn more – video tutorials about the basics of Fluorescence30 2/23/2012 | Life Technologies™ Proprietary and confidential