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1Immunofluorescence Staining
www.ptglab.com
IMMUNOFLUORESCENCE
STAINING
How To Optimize Your
Technical Tips and Troubleshooting
www.ptglab.com
2Immunofluorescence Staining
www.ptglab.com
CONTENTS
Fundamental Principles
Sample Preparation
Fixation
Autofluorescence
Visualization
Mounting Media
Controls
FAQs and Tips
Contact Us
3–4
5–6
7
8
9–10
11
12
13–15
16
3Immunofluorescence Staining
www.ptglab.com
FUNDAMENTAL PRINCIPLES
–– Widely used technique in biological 	
	 research/clinical diagnostics.
–– The location, expression and pattern
	 of the target antigen can be identified.
–– IF utilizes fluorescent-labelled 	 	
	 antibodies to detect specific
	 target antigens.
4Immunofluorescence Staining
www.ptglab.com
FUNDAMENTAL PRINCIPLES
Drawbacks Besides the many advantages of IF
staining, there are also disadvantages:
–– Fixation results in cell killing.
–– Dynamic and fast processes cannot
	 be monitored.
–– Snapshot image.
–– Fixation/permeabilization results
	 in artefacts.
–– Several controls are needed .
–– IF samples are not suitable for long
	 time storage.
5Immunofluorescence Staining
www.ptglab.com
SAMPLE PREPARATION
Cell Culture –– Up-front literature research about 	 	
	 protein localization and cell confluency.
–– Too high or too low cell density might 	
	 influence the normal cell structure.
–– Seed cells in 12- or 24 well plate
	 on coverslips.
–– Let them grow until the desired cell 	
	 density is reached.
–– If performing stimulation experiments, 	
	 recalculate cell number and density.
Note
–– Junction proteins need appropriate 	
	 confluency to represent cell–cell contacts.
6Immunofluorescence Staining
www.ptglab.com
SAMPLE PREPARATION
Working
Environment
Coating
–– Antibodies are optimized by the
	 immune system.
–– PBS: Cell surface stainings.
–– Potassium: Intracellular environment.
–– Some cell types may not attach well
	 to glass cover slides.
–– The coating matrix always depends
	 on the cell type.
–– Most cell lines attach well to
	 Poly-L-Lysine.
7Immunofluorescence Staining
www.ptglab.com
FIXATION
Most Common
Used Fixatives
Type of fixative Name Advantage Disadvantage
Organic solvent Methanol
Cellular architecture
is conserved.
Damaging several epitops.
Lipid components are
getting lost.
Organic solvent Acetone Gentle for epitops.
Lipid components are
getting lost.
Chemical cross-linker Paraformaldehyde
Cellular
morphology conserved.
Cross-linking of
epitops, autofluorescence.
Note
–– If not immediately proceeding to the staining, leave some PBS in the well and store sealed at 4°C.
8Immunofluorescence Staining
www.ptglab.com
AUTOFLUORESCENCE
Biological
Autofluorescence
Autofluorescence
Fixative-induced
Autofluorescence
–– Complicates the analysis of IF.
–– Spectra of autofluorescence is very broad.
–– Cells contain components that show fluorescence.
–– Endogenous autofluorescence mainly comes from:
	 mitochondria, lyosomes, aromatic amino acid components.
–– Aldehyde fixatives can react with cellular amines/proteins
	 to form fluorescent products.
–– Prevention: Aldehyde groups can be reduced.
Note
–– Several commercial reagents are available to optimize
	 and reduce background signal and autofluorescence.
9Immunofluorescence Staining
www.ptglab.com
VISUALIZATION
Dependent on the target of interest, direct
or indirect visualization is recommended.
Direct Visualization
Indirect
Visualization
–– Labelled primary antibody.
–– Short protocol, fast analysis,
	 easy to handle.
–– No signal amplification.
–– Not suitable for low expressed targets.
–– Labelled secondary antibody.
–– Longer protocol.
–– More flexibility.
–– Suitable for medium to high
	 expressed targets.
Factor Direct Indirect
Time Short Longer
Complex No More steps
Flexible Limits Flexible
Sensitive
Weaker
signal
Strong signal
bkg Reduced bkg
Cross-
reaction
Reduced
Cross-
reaction
10Immunofluorescence Staining
www.ptglab.com
Label Characteristic Example
Conventional 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 wavelength.
QDots
Single light source, multiplex,
narrow emission.
Covering different wavelength.
Note
–– Different labels are available for direct or indirect visualization.
VISUALIZATION
11Immunofluorescence Staining
www.ptglab.com
MOUNTING MEDIA
Mounting media are:
–– PBS/ Glycerol mixture.
Why is a mounting media required:
–– Refractive index
–– Prevent photobleaching,
	 conserve sample.
How to choose a mounting media:
–– Type of imaging: fixed-longterm/	 	
	 immediately, fluorophore, 	 	 	
	 multi-fluorophores.
–– Important factors: Toxicity, shrinking, 	
	 durability, refractive index.
12Immunofluorescence Staining
www.ptglab.com
CONTROLS
To reveal unspecific staining:
–– The unstained sample (just fixed, blocked, permeabilized) 		
	 should be analyzed to understand the autofluorescence 		
	 background 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 and appropriate labeling.
13Immunofluorescence Staining
www.ptglab.com
FAQS AND TIPS
No/Weak Staining
Potential source Recommended test
Conditions of antibody
are not optimized.
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.
Protein of interest is not
expressed in used tissue.
Run a positive control.
Damaged epitope. Over-fixation, reduce fixative step or change to another fixative.
Antibody is not suitable
for detection of protein
in its native form in IF.
Perform a test on a native Western Blot (not-denaturated).
14Immunofluorescence Staining
www.ptglab.com
FAQS AND TIPS
Unspecific Staining/
No Signal
Potential cause Recommended test
Target of interest
is a nuclear protein.
Use a permeabilization step.
Weak or no fluorescent signal.
Store fluorescent labeled antibody in the dark.
Use a direct labeled primary antibody.
Use signal enhancer.
Fading signal.
Store fluorescent labeled antibody in the dark.
Choose another mounting media.
Artefacts
Can be due to: cell culture, cell density, insufficient washing, over-fixation,
mounting issues.
15Immunofluorescence Staining
www.ptglab.com
FAQS AND TIPS
Background Staining
Potential cause Recommended test
Unspecific binding of primary/
secondary antibodies.
Run control.
Prolong blocking step.
The sample is poorly washed. Repeat or prolong washing step.
Repeat or prolong
washing step.
Incubate at 4°C.
Inappropriate fixation
causes artefacts or damages
the antigen.
Reduce fixative step.
Change fixative.
Permeabilization has damaged
the cell or protein.
Decrease or skip the permeabilization step.
The slide has dried. Always keep slide moist.
16Immunofluorescence Staining
www.ptglab.com
CONTACT US
proteintech@ptglab.com
europe@ptglab.com
service@ptglab.com
Available 24 hours via Live Chat and 9–5
(CDT) via phone.
Proteintech Group
Proteintech Europe
Proteintech
Support
US Head Office
United Kingdom
China Office
Please visit us at www.ptglab.com for
more information about our antibodies
and technical tips.

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How to Optimize Your Immunofluorescence Staining

  • 1. 1Immunofluorescence Staining www.ptglab.com IMMUNOFLUORESCENCE STAINING How To Optimize Your Technical Tips and Troubleshooting www.ptglab.com
  • 2. 2Immunofluorescence Staining www.ptglab.com CONTENTS Fundamental Principles Sample Preparation Fixation Autofluorescence Visualization Mounting Media Controls FAQs and Tips Contact Us 3–4 5–6 7 8 9–10 11 12 13–15 16
  • 3. 3Immunofluorescence Staining www.ptglab.com FUNDAMENTAL PRINCIPLES –– Widely used technique in biological research/clinical diagnostics. –– The location, expression and pattern of the target antigen can be identified. –– IF utilizes fluorescent-labelled antibodies to detect specific target antigens.
  • 4. 4Immunofluorescence Staining www.ptglab.com FUNDAMENTAL PRINCIPLES Drawbacks Besides the many advantages of IF staining, there are also disadvantages: –– Fixation results in cell killing. –– Dynamic and fast processes cannot be monitored. –– Snapshot image. –– Fixation/permeabilization results in artefacts. –– Several controls are needed . –– IF samples are not suitable for long time storage.
  • 5. 5Immunofluorescence Staining www.ptglab.com SAMPLE PREPARATION Cell Culture –– Up-front literature research about protein localization and cell confluency. –– Too high or too low cell density might influence the normal cell structure. –– Seed cells in 12- or 24 well plate on coverslips. –– Let them grow until the desired cell density is reached. –– If performing stimulation experiments, recalculate cell number and density. Note –– Junction proteins need appropriate confluency to represent cell–cell contacts.
  • 6. 6Immunofluorescence Staining www.ptglab.com SAMPLE PREPARATION Working Environment Coating –– Antibodies are optimized by the immune system. –– PBS: Cell surface stainings. –– Potassium: Intracellular environment. –– Some cell types may not attach well to glass cover slides. –– The coating matrix always depends on the cell type. –– Most cell lines attach well to Poly-L-Lysine.
  • 7. 7Immunofluorescence Staining www.ptglab.com FIXATION Most Common Used Fixatives Type of fixative Name Advantage Disadvantage Organic solvent Methanol Cellular architecture is conserved. Damaging several epitops. Lipid components are getting lost. Organic solvent Acetone Gentle for epitops. Lipid components are getting lost. Chemical cross-linker Paraformaldehyde Cellular morphology conserved. Cross-linking of epitops, autofluorescence. Note –– If not immediately proceeding to the staining, leave some PBS in the well and store sealed at 4°C.
  • 8. 8Immunofluorescence Staining www.ptglab.com AUTOFLUORESCENCE Biological Autofluorescence Autofluorescence Fixative-induced Autofluorescence –– Complicates the analysis of IF. –– Spectra of autofluorescence is very broad. –– Cells contain components that show fluorescence. –– Endogenous autofluorescence mainly comes from: mitochondria, lyosomes, aromatic amino acid components. –– Aldehyde fixatives can react with cellular amines/proteins to form fluorescent products. –– Prevention: Aldehyde groups can be reduced. Note –– Several commercial reagents are available to optimize and reduce background signal and autofluorescence.
  • 9. 9Immunofluorescence Staining www.ptglab.com VISUALIZATION Dependent on the target of interest, direct or indirect visualization is recommended. Direct Visualization Indirect Visualization –– Labelled primary antibody. –– Short protocol, fast analysis, easy to handle. –– No signal amplification. –– Not suitable for low expressed targets. –– Labelled secondary antibody. –– Longer protocol. –– More flexibility. –– Suitable for medium to high expressed targets. Factor Direct Indirect Time Short Longer Complex No More steps Flexible Limits Flexible Sensitive Weaker signal Strong signal bkg Reduced bkg Cross- reaction Reduced Cross- reaction
  • 10. 10Immunofluorescence Staining www.ptglab.com Label Characteristic Example Conventional 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 wavelength. QDots Single light source, multiplex, narrow emission. Covering different wavelength. Note –– Different labels are available for direct or indirect visualization. VISUALIZATION
  • 11. 11Immunofluorescence Staining www.ptglab.com MOUNTING MEDIA Mounting media are: –– PBS/ Glycerol mixture. Why is a mounting media required: –– Refractive index –– Prevent photobleaching, conserve sample. How to choose a mounting media: –– Type of imaging: fixed-longterm/ immediately, fluorophore, multi-fluorophores. –– Important factors: Toxicity, shrinking, durability, refractive index.
  • 12. 12Immunofluorescence Staining www.ptglab.com CONTROLS To reveal unspecific staining: –– The unstained sample (just fixed, blocked, permeabilized) should be analyzed to understand the autofluorescence background 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 and appropriate labeling.
  • 13. 13Immunofluorescence Staining www.ptglab.com FAQS AND TIPS No/Weak Staining Potential source Recommended test Conditions of antibody are not optimized. 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. Protein of interest is not expressed in used tissue. Run a positive control. Damaged epitope. Over-fixation, reduce fixative step or change to another fixative. Antibody is not suitable for detection of protein in its native form in IF. Perform a test on a native Western Blot (not-denaturated).
  • 14. 14Immunofluorescence Staining www.ptglab.com FAQS AND TIPS Unspecific Staining/ No Signal Potential cause Recommended test Target of interest is a nuclear protein. Use a permeabilization step. Weak or no fluorescent signal. Store fluorescent labeled antibody in the dark. Use a direct labeled primary antibody. Use signal enhancer. Fading signal. Store fluorescent labeled antibody in the dark. Choose another mounting media. Artefacts Can be due to: cell culture, cell density, insufficient washing, over-fixation, mounting issues.
  • 15. 15Immunofluorescence Staining www.ptglab.com FAQS AND TIPS Background Staining Potential cause Recommended test Unspecific binding of primary/ secondary antibodies. Run control. Prolong blocking step. The sample is poorly washed. Repeat or prolong washing step. Repeat or prolong washing step. Incubate at 4°C. Inappropriate fixation causes artefacts or damages the antigen. Reduce fixative step. Change fixative. Permeabilization has damaged the cell or protein. Decrease or skip the permeabilization step. The slide has dried. Always keep slide moist.
  • 16. 16Immunofluorescence Staining www.ptglab.com CONTACT US proteintech@ptglab.com europe@ptglab.com service@ptglab.com Available 24 hours via Live Chat and 9–5 (CDT) via phone. Proteintech Group Proteintech Europe Proteintech Support US Head Office United Kingdom China Office Please visit us at www.ptglab.com for more information about our antibodies and technical tips.