ICC/IF

Immunocytochemistry (ICC) is a common research technique by which a user is able to view and identify cellular components and the locations of proteins of interest in intact cells. Typically, ICC is performed using fluorescently labeled antibodies (Immunofluorescence – IF) for easier multiplexing which facilitates visualization of colocalization of proteins. However, ICC can also be performed using colorimetric reagents. 

ICC Protocol – Direct Staining

Materials

  • Tris buffered saline (TBS)
    • (50 mM Tris, 150 mM NaCl, pH 7.4) 
  • Or, Phosphate buffered saline (PBS)
    • (137 mM NaCl, 2.7 mM KCl, 10 mM Na2HPO4, 1.8 mM KH2PO4, pH 7.4)
  • Fixation reagent: 4% formaldehyde in PBS
  • Blocking reagent: 1 – 5% normal serum or BSA.
  • Primary antibody: fluorophore-conjugated format
  • Nuclear counterstain: DAPI or Hoechst
  • Mounting medium: Various mounting mediums are available.  We prefer ProLong Antifade by ThermoFisher.
  • Humidified container such a cell incubator.
  • Parafilm
  • Glass coverslips
  • Clear nail polish

Methods

  1. Cells are plated at an appropriate density and allowed to attach to the slide or dish (ex. 30,000 cells/chamber in an 8-chamber slide).
    • Cells are usually plated one day prior to staining in order to achieve 60-80% confluency.
  2. Fix the cells with 4% formaldehyde for 15 min at room temperature. (The optimal fixation time and reagent depends on the antigen of interest and must be optimized.)
  3. Gently wash the cells 3 times in PBS (5 min/wash) using a dropper to add PBS to the chamber followed by aspiration to remove the buffer.
    • Note: It is critical from this point on that the cells do not dry out as this will cause increased levels of background staining and difficulty interpreting staining results. It is best not to aspirate more than 2 wells at a time in order to reduce the possibility of the cells within the wells drying.
  4. Cover the cells with blocking solution (100 µL/chamber in an 8-chamber slide). Tightly cover the opening of the chamber slide with Parafilm. Incubate in a humidified chamber, 1 hour at room temperature.
  5. Remove the Parafilm cover and gently wash the cells 3 times in PBS or TBS (5 minutes/wash) as described in step 3.
  6. Dilute the fluorophore-conjugated antibody or multiple fluorophore-conjugated antibodies,at manufacturer’s recommended dilution in blocking reagent and overlay onto cells protecting from light. Tightly cover the opening of the chamber slide with Parafilm. Incubate in a humidified chamber overnight at 4°C.
  7. Remove the Parafilm cover and gently wash the cells 3 times in PBS or TBS (5 min/wash) as described in step 3.
  8. Optional: Nuclei can be counterstained using DAPI or Hoescht.
    • It is necessary to select a counterstaining agent, with a fluorescent emission spectra, that does not overlap with the emission spectra of the other fluorophores used in the experiment.
  9. Mount and coverslip using your preferred mounting medium with DAPI. Seal the edge of the coverglass with clear nail polish.
  10. Allow slides to dry for 1-2 hours before visualizing.
  11. Slides can be stored at 4°C protected from light if needed.