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The Complete Guide To Optimizing

IMMUNOFLUORESCENCESTAINING

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The Complete Guide To Optimizing Immunofluorescence Stainingptglab.com

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FOREWORD
Immunofluorescence (IF) staining is a widely usedtechnique in biological research and clinical diagnostics.
IF utilizes fluorescent-labeled antibodies in order to detectspecific target antigens. Followed by imaging, it is a very directtechnique as you can actually see something. Although it is awell-established tool, multiple factors have to be consideredand various optimization steps have to be taken to ensure
successful staining.
This guide provides not only an introduction toimmunofluorescence staining, but also includes protocols anddetailed troubleshooting. We discuss and present useful tips for
preparing optimal samples that produce the best signal-to-noise
ratios for immunofluorescence staining signals.

What’s Inside

6–8
General Protocols
9–10
Sample Preparation
11–12
Signal-To-Noise Ratios
13–14
Visualization
15
IF Staining Controls
16
Afterword
17–18
FAQs and Tips
19
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The Complete Guide To Optimizing Immunofluorescence Staining

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The Complete Guide To Optimizing Immunofluorescence Staining

GENERAL PROTOCOLS
Fundamental Principles

There is no one perfect protocol of how to fix, stain, andanalyze cells. Producing beautiful, eye-catching, ready-topublish IF stainings requires optimization of the best conditions
for each single step of the protocol in accordance with sample
type, target of interest, antibody used, antibody performance,
and antigen-antibody combination.
These days, microscope software (e.g., deconvolution software,etc.) is advanced. It helps to enhance the quality of the image
and can often be used to overcome the deficiencies of a poorly
prepared sample. However, attention has to be paid to thesetechniques as they might lead to misinterpretation of results.
Damaged cell structures or poor labeling of a suboptimal
prepared sample can give false data. No microscopy time should
be wasted on poorly prepared samples. Therefore, it is essential
to appropriately prepare, fix, permeabilize, block, label, andmount your sample. Still, it is common in a lot of laboratories
to use the same standardized protocol for all experiments once
it has been established.
Therefore, it is important to understand the basics of an IF
staining protocol and to know about the variation that could beimplemented. Together with an up-front literature research aboutcell type, antigen, and antibody, you can save time and get closer
to generating a beautiful staining for your research.

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The Main Steps of IF
Overview of the main stepsof IF.

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The Complete Guide To Optimizing Immunofluorescence Staining

IF Staining Protocols

Please note: This is a general protocol that provides a good startfor testing a new sample, target, or antibody. The protocol has
been developed by Proteintech’s R&D Team. It may, however,require further adaptation for each specific experiment.
Fixation and Permeabilization
1.

Aspirate medium, then briefly wash cells seeded on clean
glass coverslips with 1X PBS.

2.

Fix the cells for 10–20 min. Rinse coverslip with 1X PBS
3 times for 3 min each.

3.

Treat coverslip with permeabilization buffer (e.g. 		
TritonX-100, 0.1 to 1% in PBS) for 10–20 min at room
temperature. Rinse coverslip 3 times with 1X PBS for
3 min each.

Blocking
4.

Prepare blocking solution. Incubate the cells with blocking
solution at room temperature for 1 h.

Primary & Secondary Antibody Incubation
5.

Aspirate blocking solution, apply primary antibody
diluted in antibody dilution buffer. At the same time,
set up a negative control without applying the primary
antibody. Incubate for 1 to 2 h at RT or overnight (ON)at 4°C in the dark.

6.

Wash coverslip with 1X PBS 3 times for 3 min each.

7.

Apply corresponding fluorochrome-labeled secondary
antibody diluted in antibody dilution buffer and incubate
for 1 h at room temperature in a moist environment inthe dark.

8.

Wash coverslip with 1X PBS 3 times for 3 min each.

Mounting and Visualization
9.

Mount coverslip. Optional counterstain with DAPI fornuclei detection.

10. Examine slides at a microscope.

Solutions Needed
1X PBS

1000 ml

Antibody dilution buffer

100 ml10 mM Na₂HPO₄

1.42 g

1% BSA1g

1.8 mM KH₂PO₄

0.24 gAdd 1X PBS to 100 ml–137 mM NaCl

8g––

2.7 mM KCl

0.2 g––––

Adjust pH to 7.4; Add ddH₂O to 1000 ml

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9SAMPLE PREPARATION
Cell Culture

To get the best staining pattern, it is worth conducting anup-front literature research about protein localization andcell confluency.
At time of staining, the cell density should be around 50%.
When cell density is too high, cell architecture is deformed
and may effect higher background at low magnifications.
When cell density is too low, it is more challenging to findthe field with the optimal cell pattern.
1.

Seed cells in appropriate volume of media in 12- or 24well plates on coverslips.

2.

Let them grow until the desired cell density is reached.

3.

If you are performing any stimulation experiments, 		recalculate cell number and time to reach cell density
needed after the stimulation experiment.

Tip: Know your protein of interest. For instance, stainings forjunction proteins need the appropriate cell confluency inorder to represent the cell–cell contacts. Different celldensities might show different results here.

Coverslip Preparation

1.

Sterilize coverslips using an autoclave.

2.

Wash coverslips briefly with ethanol in a laminar flow hood.

3.

Let coverslips dry completely before use.

4.

Add coverslips to the well plate using tweezers.

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CoatingThe Complete Guide To Optimizing Immunofluorescence Staining

Some cell types may not attach well to glass coverslips.
Special Coating Circumstances

Fixation•

Non-adherent and weakly attached cells are difficult togrow on glass surfaces. In this case, coat the coverslips
with poly-lysine or extracellular matrices (e.g., collagenor laminin).

•Some tissues (e.g., blood vessels or brain samples) may
require gelatin-coated or poly-lysine-coated slides toremain attached to the slide after multiple wash steps.

1.

Add an appropriate volume of coating solution to thecoverslip, covering the whole surface. Rock the well platesmoothly in order to ensure equal coating of the surface.

2.

Incubate the coating solution according to the manufacturer’s
instructions (time, temperature).

3.

Thoroughly wash the surface with 1x PBS or sterile cell
culture grade water.

1.

When cells have reached the desired cell confluency,
aspirate the cell culture media and wash 2x PBS.

2.

Add fixative to each well, making sure the surface is wellcovered. Incubation time depends on the fixative used.

3.

Remove the fixative and wash 2x PBS.

4.

If not immediately proceeding to antibody staining,leave some PBS in the well and store the sealed plateat 4°C. Fixated cells can be stored for a few weeks inthe cold. Make sure the coverslips don’t dry out.

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SIGNAL-TO-NOISE RATIOS
Autofluorescence

Autofluorescence complicates the analysis of IF stainings.
In general, the spectra of autofluorescence are very broad,compared to those of fluorescent probes. Thus, it is challenging
to distinguish between autofluorescence and the actual
fluorescence of interest, leading to misunderstanding of theimage analysis. To overcome the problem of autofluorescence,
it is important to have an idea of its source before startingto optimize the protocol or playing around with the filter
sets. Autofluorescence is often used as a collective term for“biological autofluorescence” or so-called “fixativeinduced autofluorescence.”

Biological
Autofluorescence

Cells contain components that show fluorescent when excitedby a suitable wavelength. This biological autofluorescence has its
origin in endogenous fluorophores. Biological autofluorescence
mainly comes from mitochondria, lysosomes, and aromatic aminoacid components. The most important components causing
intrinsic fluorescence are Flavin coenzymes (FAD, FMN) (Figure 1)
or pyridine nucleotides (NADH) (Figure 2).

Figure 1
Structural formula of flavinadenine dinucleotide (FAD),
showing absorption at 450 nm
and emission at 515 nm.

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The Complete Guide To Optimizing Immunofluorescence Staining

Figure 2
Structural formula ofNicotinamide adenine
dinucleotide (NADH),
showing absorption at 340 nm
and emission at 460 nm.

Fixative-induced
Autofluorescence

When fixing with aldehydes, these aldehydes can react withcellular amines or proteins, resulting in a fluorescent product.
These issues can be diminished by reducing the aldehyde group
to a hydroxyl group (e.g., by sodium borohydride).
Please note: These days, several commercial reagentsare available to optimize and reduce background signal
and autofluorescence. There are ready-to-use drop-in solutions
on the market that suppress background signal and cell culture
media suitable for live cell imaging. In case of a backgroundsuppressor that chemically reduces autofluorescence, overdosage has to be avoided as it is always combined with a lossof the actual signal.

Most Commonly Used Fixative For IF Staining
Type of fixative

Name

Advantage

Organic solvent

Methanol

Cellular architecture
is conserved.

Organic solvent
Chemical Cross-linker

Disadvantage
Damaging toseveral epitopes.
Lipid components lost.

AcetoneGentle for epitopes.

Lipid components lost.

Paraformaldehyde

Cellular morphology
conserved.

Cross-linking of epitopes,autofluorescence.

ptglab.com13VISUALIZATION
Depending on the target of interest, direct or indirect
visualization is recommended.

Direct Visualization

Indirect Visualization

Direct visualization requires the use of a labeledprimary antibody:•

Short protocol, fast analysis, easy to handle.

•Expensive, a labeled primary antibody for eachtarget is needed.

•

A highly specific primary antibody is needed.

•

No signal amplification, not suitable for low
expressed targets.

Indirect visualization requires the use of a labeledsecondary antibody:•Longer protocol.

•Less expensive, a labeled secondary antibody canbe used for different primary antibodies.

•Higher background noise.

•Signal amplification, suitable for medium to highexpressed targets.

Direct Vs. Indirect Immunofluorescence
DirectIndirectTarget Structure

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The Complete Guide To Optimizing Immunofluorescence Staining

Most Commonly Used Labels For IF Staining
Label

Characteristic

ExampleConventional fluorescent labels

Medium bright and photostable,
replicate historic experiments

FITC, R-PE, TRITC, Cy3

Alexa® labels, DyLight

Cover the white range, common
filter sets, bright, photostable, not
pH sensitive

Covering different wavelengths

QDotsSingle light source, multiplex,
narrow emission

Covering different wavelengths

Mounting Media

Since a fluorescent signal fades away during time, mounting
media is added at the end of the staining protocol to maintainthe sample. It is also needed to optimize the refractive index for
imaging. Mounting media are a solution of glycerol in a specialbuffer. This helps to maintain the fluorescence signal andslow down photobleaching. Mounting media are optimized
for different conditions and dyes, and some questions toconsider when selecting the right one are:•Fixed samples for long-term conservation?

•Fixed samples for immediate imaging?

•Which kind of dye/fluorophore?

•Multistaining?

Tip: Poorly prepared samples show artifacts that could be
misinterpreted. For instance, bubbles, insufficient washing,over-incubation with antibodies, or over-fixation can resultin unspecific staining patterns and show false-positiveresults. Adding too much or too little mounting media
can create artifacts and damage the sample.

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IF STAINING CONTROLSIn order to understand and interpret the IF images obtained,different control stainings can be performed that help to reveal
unspecific staining.
•The unstained sample (just fixed, blocked, permeabilized)
should be analyzed to understand the autofluorescencebackground signal.

•A sample (just fixed, blocked, permeabilized) that is
incubated with the secondary antibody reveals whether
the secondary antibody binding is specific.

•To ensure specific binding of the primary antibody,the sample can be blocked with a specific blocking peptide(used to raise the primary antibody). Binding of the primary
antibody will be inhibited.

•In case of multistainings, the staining should also be
performed separately to ensure no cross-reactions andappropriate labeling.

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The Complete Guide To Optimizing Immunofluorescence Staining

AFTERWORD
As mentioned at the beginning of this guide, IF staining is a
powerful tool with many benefits when used in/for analysis of atarget of interest. Conversely, there are certain drawbacks about
which you should be aware:•The fixation step results in the killing of cells and dynamic
and fast processes cannot be monitored. An IF image is asnapshot of the cell at a certain point in time.

•Fixation or permeabilization also result in artifacts. In orderto ensure that no artifacts are observed, it is necessary toperform a couple of additional time-consuming controls.

•The storage time for IF samples is short due tophotobleaching and the limited stability of thefluorescent label.

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17FAQs AND TIPS
No/Weak Staining

Potential cause

Conditions of antibody
are not optimized.

Non-specific Staining/No Signal

Recommended test
Titrate the antibody
concentration to optimize
best working conditions.
Incubate the primary
antibody at room temperature
or at 4°C overnight.

Protein of interest is low
expressed in used cells.

Use signal amplification
when visualizing.

Damaged epitope.

Over-fixation, reduce
fixative step, or change
to another fixative.

Antibody is not suitablefor detection of protein
in its native form in IF.

Perform a test on anative Western Blot
(not-denaturated).

Potential cause

Recommended test

Target of interest is anuclear protein.

Use a permeabilization step.
Store fluorescent-labeled
antibody in the dark.

Weak or no fluorescent signal.

Use a direct-labeledprimary antibody.
Use signal enhancer.

Fading signal.

Artifacts.

Store fluorescent-labeled
antibody in the dark.
Choose anothermounting media.
Can be due to: cell culture,cell density, insufficientwashing, over-fixation,
mounting issues.

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Background Staining

The Complete Guide To Optimizing Immunofluorescence Staining

Potential cause

Recommended test

Non-specificbinding of primary/secondary antibodies.

Run control.
Prolong blocking step.

The sample is poorly washed.

Repeat or prolong
washing step.

The antibody incubation
temperature is too high.

Incubate at 4°C.

Inappropriate fixation
causes artifacts or damages
the antigen.

Reduce fixative step.

Permeabilization has
damaged the cell or protein.

Decrease or skip thepermeabilization step.

The slide has dried.

Always keep slide moist.

Change fixative.

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