ICC is used to understand the distribution and localization of proteins within compartments of a cell. ICC detects specific antigens in preserved cell populations using an appropriate antibody labeling strategy. Samples are collected, fixed to maintain cell morphology, architecture and the antigenicity of target epitopes. A variety of antibody staining schemes can produce informative ICC images. Having an antibody conjugated to a fluorophore (immunofluorescence) is a common detection method. See our validation data page for immunocytochemical data. Click for related immunohistochemistry protocol.
Depositing/Fixing the cells:
It is recommended that the cells are grown on 100-mm or 150-mm diameter tissue culture dishes and that the cells are then seeded on 6-well tissue culture plates before the day of the experiment. Seed adherent cells on 6-well tissue culture plates in a sterile tissue culture hood.
- Sterilize glass coverslips by dipping them in 90% ethanol and carefully drying them over a flame for a few seconds.
- Place each coverslip in sterile 6-well tissue culture plates.
- Add 1-2ml of cell suspension over each coverslip in the 6-well plates.
- Grow the cells at 37°C in a humidified CO2 incubator until they are 50-70% confluent.
- Aspirate the culture medium from each well and gently rinse the cells twice in PBS at room temperature. Do not let the cells dry out.
- Fix the cells by incubating them in 4% (v/v) paraformaldehyde in PBS for 20 minutes at room temperature.
- Rinse the cells three times with PBS. The cells can be stored in 0.02% (w/v) sodium azide in PBS at 4°C for several days.
- Proceed to Antigen Retrieval Protocol, if necessary.
Note: Some antibodies work best if the cells are heated in Antigen Retrieval Buffer (HIER).
Permeabilize / Staining Procedure:
- Incubate the cells in 0.1% Triton X-100 in PBS for 15 minutes at room temperature.
- Rinse the cells 3 times in PBS.
- Incubate the cells in 10% goat serum in PBS for 1 hour at room temperature.
- Dilute the primary monoclonal antibody/antibodies to the appropriate concentration using block solution; the final volume should be sufficient to cover each coverslip (e.g. 0.5 ml-1 ml per coverslip).
- Incubate the cells in the primary antibody/antibodies at 4°C, overnight, or at room temperature for 2 hours.
Note: When using fluorophore-conjugated secondary antibodies, proper controls are critical to obtaining informative images.
a) Carry out the ICC protocol without adding the primary antibody and the secondary antibody. Any fluorescence seen is due to autofluorescence of the sample.
b) Carry out the ICC protocol without adding the primary antibody only. Any fluorescence seen is due to the secondary antibody binding nonspecifically to the sample.
- Rinse the cells in 1% goat serum in PBS 3 times for 10 minutes.
- Dilute the fluorophore-conjugated secondary antibody/antibodies, away from light, in 10% block solution. Be sure that the correct isotype-specific secondary antibody for each primary antibody is used.
- Incubate the cells in the fluorophore-conjugated secondary antibodies for 2 hours at room temperature, away from light.
- Rinse cells in 1% block solution for 3x10 minutes, away from light.
- Dilute DAPI to 300 ng/mL in 1% block solution. Incubate the cells for 10 minutes in the diluted DAPI, away from light.
- Label a microscope slide for each coverslip.
- Add a drop of mounting medium to each slide.
- Pick up each coverslip with a forceps and place it on the mounting medium, with the cell-side face down.
- Apply nail polish or glue along the edges of the coverslips to seal them to the slides.
- Visualize the cells using a fluorescence microscope equipped with the appropriate filters for DAPI dye, Texas Red dye and Fluorescein isothiocyanate dye.
For more information, see: