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Troubleshooting of Immunofluorescence

Immunofluorescence (IF) staining is a widely used technique in biological research and clinical diagnostics. Here Creative Biolabs lists the common IF troubleshooting tips. Though the tips provided here cover many different problems you may encounter in IF, we hope that you will find the information beneficial to you and useful as a reference guide. If you ever need more assistance with your experiments, please contact us for additional help.

Protocol of Immunofluorescence is available for you.

Contents

Weak or No Staining

Possible Cause Recommended Solution
Incorrect light source/filter set Make sure your microscope is equipped with the correct light source/ filter set for the fluorophore you have chosen.
Gain/exposure is too low Turn up the gain and/or increase the exposure time.
Fluorescent tag bleached Avoid over exposure of the slide to light sources for extended periods. Always store slides in the dark.
Samples are over fixed
  • Perform antigen retrieval to unmask the epitope.
  • Reduced the duration of fixation.
Cells are not permeabilized
  • Methanol and acetone fixation methods are available.
  • For formaldehyde, permeabilize cells with 0.2% Triton X-100.
Tissue/cells dried out Samples must be kept covered in liquid throughout the staining process.
Not enough primary antibody Use a higher concentration of antibody.
The primary and secondary antibodies are incompatible
  • The secondary antibody should be raised against the host of the primary antibody.
  • Isotypes should be compatible.
Slide storage Samples should be imaged shortly after processing as the signal decreases over time. Store slides at 4⁰C in the dark if needed.
Antibody storage Antibody was not stored as recommended. A new vial will need to be used if necessary.
Low expression of protein of interest
  • Run a positive control.
  • Use an amplification step to maximize the signal.
  • Pair with a brighter fluorophore.
Incubation time is too short Increase the incubation time.
Inappropriate testing model Protein expression should be confirmed by WB or other means.
Target protein not induced properly Optimal treatment conditions and controls should be determined for each target.

Non-Specific Staining

Possible Cause Recommended Solution
Spectral overlap Adjust your light sources/filters to pick up only one signal at a time. If this is not possible, choose new fluorophores that do not have spectral overlap.
Antibody concentration is too high Reduce the concentration, and the incubation period.
Aggregates Spin down secondary antibodies in a microcentrifuge to move aggregates to the bottom of the tube.
The primary antibody is raised again the same species as the tissues stained
  • Try using a primary that is raised against a different species.
  • Try to block the endogenous IgG with serum from the same species as the secondary.
  • Incubate sections with 1% Triton to clean the tissues. Or Use TBS-Tween 20 as a washing buffer instead of PBS-Tween 20.

High Background

Possible Cause Recommended Solution
Sample autofluorescence
  • Detect the auto-fluorescence before staining and operate the fluorescence quenching if there is auto-fluorescence.
  • Set negative control and decrease the parameters of the fluorescence microscope to reduce background.
  • Try to avoid using samples with high concentration of riboflavin and cytochrome and other cell components with auto-fluorescence.
  • Avoid cell death.
Insufficient blocking Use normal serum from the same species as the secondary antibody used.
Incorrect antibody dilution Dilute according to recommended antibody dilution.
Samples dried out Samples must be kept covered in liquid throughout the staining process.
Insufficient washing Increase washing times.
Antibody cross-reactivity Use isotype control antibodies to determine whether your secondary antibody is cross-reacting.
Non-specific antibody binding Compare to knockdown or knockout cells or compare to cells known to express higher or lower levels of the target antigen.

Negative Control Stains Positive and/or Positive Control Stains Negative

Possible Cause Recommended Solution
Use of wrong control or reagents Check reagent labels and repeat test.
Cross-contamination of controls and/or specimens
  • Always change pipettes or pipettor tips between use of controls and specimens.
  • Always return proper caps to reagent vials and specimen containers.
Running of controls and/or specimens between wells Use smaller drops to stay within well perimeters.
Splashing of excess control and/or specimens from well to well due to over vigorous rinsing with wash bottle
  • Follow rinse directions carefully.
  • Shake off excess reagent and gently agitate slide in jar of fresh PBS.

Cells Visible but Negative Staining with All Tests Including Positive Control

Possible Cause Recommended Solution
Reagents used in wrong sequence or one or more reagents not added Review procedure and repeat test following staining steps precisely.

More Positive Results than Expected (False Positives) with IgM Testing

Possible Cause Recommended Solution
Presence of rheumatoid factor Pretreatment of serum, using ion exchange chromatography or IgG immunoprecipitation, to remove IgG interference.

More Negative Results than Expected (False Negatives) with IgM Testing

Possible Cause Recommended Solution
Competition from IgG specific antibodies. Pretreatment of serum, using ion exchange chromatography or IgG immunoprecipitation, to remove IgG interference.

Weaker Results than Expected with IgM Testing

Possible Cause Recommended Solution
Incubation time too short Increase the incubation time.
Inadequate incubation temperature Incubate at 37°C.

Results Differ from Those Reported by Another Laboratory

Possible Cause Recommended Solution
Microscope optics, filters, light sources, reagents and personnel may vary enough to result in differences of twofold or more. Each laboratory must establish their own normal ranges. Results between laboratories should not be compared.

Notes for Fluorescent Double Staining

For Direct Method:

  1. The species of primary antibodies are not specially required, and the fluorescent labels of two primary antibodies should be different.

For Indirect Method:

  1. It is suggested to use primary antibodies from two different species, and secondary antibodies with different fluorescence labeling.
  2. It is suggested to incubate one primary antibody, then incubate the fluorescence secondary antibody which matches the primary antibody, then followed with incubation of another primary antibody and its matched fluorescence secondary antibody.
  3. Set positive control and negative control.
  4. Use blocking serum that was collected from the species in which the secondary antibody was raised.

The other procedures are the same as conventional IF experiments.