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

First identify the problem with your immunocytochemistry staining from the options below:

Weak or No Staining

Cells have detached from coverslip:

• Reduce washing steps or wash less vigorously
• Adhere cells to the coverslip using poly-lysine

Cells were not permeabilized:

• Methanol and acetone fixation will permeabilize cells
• If using formaldehyde, permeabilize cells with 0.2% Triton X-100

Cells dried out:

• Samples must be kept covered in liquid throughout the staining process

Cell/tissues are over fixed:

• Reduce the duration of fixation

Not enough primary antibody:

• Use a higher concentration of antibody
• Incubate longer

Incubation time is too short:

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

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.

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 ICC
• Test the antibody in a western blot to make sure it hasn’t been damaged.

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.

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

 

High Background

Antibody Concentration is too high:

• Dilute the primary and/or secondary antibody further

Non-specific binding:

• Run a secondary antibody control without the primary antibody. If there is staining, then change the secondary antibody, or use a conjugated primary antibody instead

Blocking insufficient:

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

Over amplified:

• Reduce amplification incubation time, and dilute the secondary antibody further

Insufficient washing:

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

 

Non-specific Staining

Antibody Concentrations too high:

• Try reducing the concentration, and the incubation period

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.

 

Tried everything from our immunocytochemistry troubleshooting guide and still having trouble? Check out these other useful immunostaining resources:

• Immunocytochemistry Protocol
• Immunofluorescence Troubleshooting Guide
• Immunohistochemistry Troubleshooting Guide