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Immunocytochemistry Troubleshooting

The following troubleshooting guide is intended to explain causes and possible solutions for common problems observed in immunocytochemistry/immunofluorescence (ICC/IF) staining.

No Signal

Antibody Application

Increase the concentration or incubation time of the primary or secondary antibody.

Permeabilization Buffer

  • Use the proper permeabilization reagent for the target protein’s localization. Triton detergent is necessary for mitochondrial or nuclear proteins, but will dissolve the outer membrane and disrupt proper membrane localization.
  • Increase incubation duration or detergent concentration.

Cell Fixation

  • Over fixation can cause epitope masking. Decrease the time or concentration of the fixative.

Antibody compatibility

  • Confirm that your primary and secondary antibodies are compatible by checking the species reactivity.
  • Confirm the antibody can be used for assays in which the protein is in its native conformation.
  • Ensure that the secondary is working and compatible with your microscope’s filter sets by using a positive control primary.

Target availability

  • Use an overexpression assay or positive control cell line known to express the protein of interest.

Cells Drying

  • Fluorescent signal will be lost if the cells are allowed to dry. Ring coverslips with nail polish.

Cell Viability

  • Confirm cell viability before starting the staining procedure.

Microscope Adjustments

  • Increase the exposure time of your camera.



Antibody Concentration

  • Decrease the concentration of the primary/secondary antibody.


  • Increase the incubation time or concentration of serum in the blocking buffer. Use blocking buffer for primary and secondary antibody dilutions.

Antibody Application

  • Always incubate primary antibodies overnight at 4° C. Room temperature incubation increases unspecific binding and causes higher background.
  • Confirm that the secondary is not cross-reacting with the cells by performing the assay without the primary.

Contamination Artifacts

  • Ensure slides are clean and free of dust. Buffers should be made fresh to prevent microbial contamination.


  • Increase the amount of washes. Add very gentle agitation to the plates.
  • Increase the concentration of Tween in the PBS-T.

Spectral Overlap

  • If double or triple labeling the cells, confirm that the secondaries do not overlap into the same spectral range