An Introduction to Immunofluorescent Staining of Cultured Cells

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

Learn more – video tutorials about the basics of Fluorescence

http://find.lifetechnologies.com/floid/floidtutorials/sldshr





The Beauty of High Quality Fluorescence Images







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







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







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







Observe the Cells

Live cells should be healthy and of an appropriate confluency

~50–75% confluency

Evenly distributed across the coverslip

Expected morphology







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







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







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







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







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







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







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







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







The Alexa Fluor® DyesAlexa Fluor dyes provide bright and photostableconjugates that match most filter and illumination source configurations.







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







Counterstaining

Counterstaining is useful to orient observed immunofluorescence staining

Common counterstains

Nucleic acid stains

Actin stains







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







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







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







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







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







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







Examples of Autofluorescence

Autofluorescence caused by aldehyde fixation visible through an assortment of filters







Example of Secondary-Caused Problems

Too much antibody Correct antibody titer

Too much secondary can generate overexposed images and increased on-cell background







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







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







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







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


mailto:[email protected]





An Introduction to Immunofluorescent Staining of Cultured Cells

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Transcript of An Introduction to Immunofluorescent Staining of Cultured Cells