Follow our immunofluorescence troubleshooting guide to quickly identify and get solutions for common protocol issues.

Identify the problem with your immunofluorescence staining from the options below:

Weak or No Staining

High Background

Non-specific Staining

Weak or No Staining

Incorrect light source/filter set:

• Ensure your microscope is equipped with the correct light source and filter set for the fluorophore you have chosen.

Gain/exposure is too low:

• Turn up the gain and/or increase the exposure time to ensure you are capturing any signal present.

Fluorescent tag bleached:

• Avoid over exposure of the slide to light sources for extended periods. Always store slides in the dark.

Cell/tissues are over fixed:

• Reduced the duration of fixation.
• Perform antigen retrieval to unmask the epitope.

Cells were not permeabilized:

• Methanol and acetone fixation will permeabilize cells.
• If using 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.
• Incubate longer.

The primary and secondary antibodies are incompatible:

• The secondary antibody should be raised against the host of the primary antibody. For example, if the primary antibody is a Mouse Anti-HSP70, use an Anti-Mouse secondary antibody (ie. Goat Anti-Mouse).
• Isotypes should also be compatible.

Suitability of the primary antibody:

• Confirm that the antibody has been validated in IHC, and specifically what type- formalin fixed, paraffin-embedded, fresh frozen, etc.
• Test the antibody in a western blot to make sure it hasn’t been damaged.

Slide storage issues:

• Samples should be imaged shortly after processing as the signal decreases over time. Store slides at 4⁰C in the dark if needed.

Antibody Storage issues:

• Freeze/thaw cycles are detrimental and can cause degradation. It is best to create aliquots of smaller amounts as soon as the product arrives at your location.
• Antibody was not stored as recommended. Unfortunately this might require a new vial to be used instead.
• If the secondary was not stored in the dark (when using immunofluorescence), a new vial will need to be used instead.

The protein is not present in the tissues being tested:

• Run a positive control.
• If the protein is present, but not abundantly, use an amplification step to maximize the signal.

Incubation time is too short:

• Increase the duration of incubation of the primary antibody with the sample.

 

High Background

Autofluorescence:

• Check to see if there is any fluorescence in an unstained section of the processed tissue. If there is, then this is autofluorescence in the tissue.
• Avoid glutaraldehyde fixative or wash with 0.1% sodium borohydride in PBS to remove free aldehyde groups.
• May be due to endogenous molecules (FAD, FMN, NADH, lipofuscin), and will require pre-photobleaching, treatment with sudan black, or cupric sulfate.

Tissue is too thick:

• Consider using thinner tissue sections.

Antibody Concentration is too high:

• Reduce the concentration of the primary and/or secondary antibody used.

Secondary is binding non-specifically:

• Run a secondary control without the primary. If there is staining, then change the secondary.

Blocking is insufficient:

• Increase the blocking incubation period and consider changing the blocking agent.

Amplification:

• Reduce amplification incubation time and dilute the secondary antibody.

Insufficient washing:

• Proper washing of the tissue between steps is critical. Ensure you are following the protocol guidelines for wash steps.

 

Non-specific Staining

Spectral overlap:

• If imaging more than one fluorescent probe, the fluorophores may have excitation and emission spectra that overlap. Adjust your light sources and 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 Concentrations too high:

• Try reducing the concentration, and the incubation period.

The primary is raised again the same species as the tissues stained (eg. Mouse on mouse):

• Try using a primary that is raised against a different species. Otherwise try to block the endogenous IgG with serum from the same species as the secondary. You can also try to incubate sections with 1% Triton to clean the tissues. Or Use TBS-Tween 20 as a washing buffer, rather than using PBS-Tween 20.

Aggregates:

• Spin down secondary antibodies in a microcentrifuge to move aggregates to the bottom of the tube. Take from the top.