Immunocytochemistry Troubleshooting

Symptom

Issue

Recommendations

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 crossreacting 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

 

More Resources

Immunocytochemistry Protocol