Immunocytochemistry Troubleshooting

Immunocytochemistry (ICC) handbook

ICC application resources

Multicolor ICC/IF protocol

Fluorochromes selection for multicolor ICC/IF

Sample preparation for ICC/IF experiments

Fixation & permeabilization

Blocking for ICC-IF assay

Antibody selection in ICC/IF

Detection methods for ICC IF

Controls for ICC/IF experiments

Counterstaining & mounting

Organelle markers guide

Secondary antibody handbook

View all protocols

TROUBLESHOOTING

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.

Background

Antibody Concentration

• Decrease the concentration of the primary/secondary antibody.

Blocking

• 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.

Washing

• 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