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
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The following troubleshooting guide is intended to explain causes and possible solutions for common problems observed in immunocytochemistry/immunofluorescence (ICC/IF) staining.
Increase the concentration or incubation time of the primary or secondary antibody.
- 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.
- Over fixation can cause epitope masking. Decrease the time or concentration of the fixative.
- 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.
- Use an overexpression assay or positive control cell line known to express the protein of interest.
- Fluorescent signal will be lost if the cells are allowed to dry. Ring coverslips with nail polish.
- Confirm cell viability before starting the staining procedure.
- Increase the exposure time of your camera.
- 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.
- 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.
- 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.
- If double or triple labeling the cells, confirm that the secondaries do not overlap into the same spectral range
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