An Introduction to Immunofluorescent Staining of Cultured Cells

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 Fluorescence

1 2/23/2012 | Life Technologies™ Proprietary and confidential

The Beauty of High Quality Fluorescence Images

2
Learn more – video tutorials about the basics of Fluorescence 2/23/2012 | Life Technologies™ Proprietary and confidential

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

3

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

4

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

5

Observe the Cells

Live cells should be healthy
and of an appropriate
confluency
 ~50–75% confluency
 Evenly distributed across
the coverslip
 Expected morphology

6

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

7

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

8

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

9

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

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 Cells blocked with BlockAid™,
appropriate serum then stained with an anti-
mitochondrial antibody and
 BlockAid™ blocking counterstained with green-
fluorescent phalloidin and
solution (B10710) DAPI

11

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

12

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 antibody
13

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

14

The Alexa Fluor® Dyes
Alexa Fluor dyes provide bright and photostable
conjugates that match most filter and illumination
source configurations.

15

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 filters

16

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

17

Counterstaining

Counterstaining is useful
to orient observed
immunofluorescence
staining

Common counterstains
 Nucleic acid stains
 Actin stains

18

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

19

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

20

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

21

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

22

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

23

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

24

Examples of Autofluorescence

Autofluorescence caused by aldehyde fixation visible
through an assortment of filters

25

Example of Secondary-Caused Problems

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

Too much antibody Correct antibody titer

26

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

27

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 urea

28

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 DAPI

29

Contact Technical Support
We 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 DAPI
Invitrogen Canada - (800) 263 6236
Learn more – video tutorials about the basics of Fluorescence

30 2/23/2012 | Life Technologies™ Proprietary and confidential

An Introduction to Immunofluorescent Staining of Cultured Cells

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