Immunocytochemistry Troubleshooting
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Symptom
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Issue
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Recommendations
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No signal
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Antibody Application
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Increase the concentration or incubation time of the primary or secondary antibody.
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Permeabilization Buffer
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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.
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Increase incubation duration or detergent concentration.
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Cell Fixation
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Over fixation can cause epitope masking. Decrease the time or concentration of the fixative.
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Antibody compatibility
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Confirm that your primary and secondary antibodies are compatible by checking the species reactivity.
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Confirm the antibody can be used for assays in which the protein is in its native conformation.
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Ensure that the secondary is working and compatible with your microscope’s filter sets by using a positive control primary.
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Target availability
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Use an overexpression assay or positive control cell line known to express the protein of interest.
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Cells Drying
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Fluorescent signal will be lost if the cells are allowed to dry. Ring coverslips with nail polish.
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Cell Viability
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Confirm cell viability before starting the staining procedure.
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Microscope Adjustments
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Increase the exposure time of your camera.
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Background
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Antibody Concentration
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Decrease the concentration of the primary/secondary antibody.
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Blocking
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Increase the incubation time or concentration of serum in the blocking buffer. Use blocking buffer for primary and secondary antibody dilutions.
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Antibody Application
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Always incubate primary antibodies overnight at 4° C. Room temperature incubation increases unspecific binding and causes higher background.
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Confirm that the secondary is not crossreacting with the cells by performing the assay without the primary.
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Contamination Artifacts
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Ensure slides are clean and free of dust. Buffers should be made fresh to prevent microbial contamination.
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Washing
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Increase the amount of washes. Add very gentle agitation to the plates.
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Increase the concentration of Tween in the PBS-T.
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Spectral Overlap
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If double or triple labeling the cells, confirm that the secondaries do not overlap into the same spectral range
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More Resources
Immunocytochemistry Protocol
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