an introduction to immunofluorescent staining of cultured cells
DESCRIPTION
This presentation covers materials, common variations and necessary controls in a immunofluorescent staining protocol and a simple guide for troubleshooting.TRANSCRIPT
1 2/23/2012 | Life Technologies™ Proprietary and confidential
An Introduction to ImmunofluorescentStaining 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
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The Beauty of High Quality Fluorescence Images
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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 images
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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. Mount
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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® Gold
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Observe the Cells
Live cells should be healthy and of an appropriate confluency
~50–75% confluency
Evenly distributed across the coverslip
Expected morphology
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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
− Acetone
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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 applications
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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 -20ºC
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Why Use Image-iT™ FX Signal Enhancer
Fluorophores can interact with structures in the cell
Image-iT™ FX signal enhancer blocks this type of background.
UntreatedImage-iT FX™ Signal
Enhancer Treated
Tetramethylrhodamine staining
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Blocking Reagent
Blocking 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 appropriate serum
BlockAid™ blocking solution (B10710)
Cells blocked with BlockAid™, then stained with an anti-mitochondrial antibody and counterstained with green-fluorescent phalloidin and DAPI
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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 counterstain
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Choosing a Secondary Antibody
Considerations:
Specificity for the primary antibody species
Avoid same-species staining
Fluorophore label must match equipment
Subtype of primary antibody
Type Pros Cons
Whole IgGStrongest signal,
inexpensivePossible crossreactivity
F(ab)2Would not interact with
F(c) receptorsLower signal due to fewer
dye molecules
Highly Cross adsorbed Less crossreactive May require higher titer
Subtype Specific IgG1, IgG2a, IgG2b, IgG3, IgMExpensive, must know
subtype of primary
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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-Red
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The Alexa Fluor® DyesAlexa Fluor dyes provide bright and photostableconjugates that match most filter and illumination source configurations.
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Why Use Alexa Fluor® Dyes?
Alexa Fluor® dyes are superior for microscopy
Very bright dyes
Photostable
Less pH-sensitive than classic dyes
Matched to common filters
Fluorescein Alexa Fluor® 488
Photobleaching Demonstration
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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 chamber
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Counterstaining
Counterstaining is useful to orient observed immunofluorescence staining
Common counterstains
Nucleic acid stains
Actin stains
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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 antifade
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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 signal
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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 647
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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 antibody
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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 antibody
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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 filters
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Examples of Autofluorescence
Autofluorescence caused by aldehyde fixation visible through an assortment of filters
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Example of Secondary-Caused Problems
Too much antibody Correct antibody titer
Too much secondary can generate overexposed images and increased on-cell background
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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 counterstaining
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Antigen Retrieval
No antigen retrieval Antigen retrieval in urea
Some antibodies require additional steps to make the antigens available for detection- KNOW YOUR PRIMARY
Staining with an antigen-retrieval–sensitive mitochondrial antibody
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Conclusion
Be patient
Every staining experiment requires optimization
Run controls with every experiment
If problems still exist, please contact our technical support scientists
FluoCells® prepared slide #2 BPAE cells stained with anti-tubulin primary and BODIPY® FL secondary antibodies,
Texas Red®-X phalloidin and DAPI
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Contact Technical Support
U.S. Technical SupportHours: 9:00 AM - 8:00 PM ESTPhone: (800) 955-6288, Option 5FAX: (800) 352-1468Email: [email protected]
Invitrogen Canada - (800) 263 6236
We succeed when you succeed!
BPAE cells stained with Alexa Fluor 488 phalloidin and DAPI
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