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Standard Protocol for Immunocytochemistry of PFA Fixed Cultured Cells

Important: Some proteins have special requirements for good detection. Please refer to the remarks sections for ICC on the respective data sheet.

Solutions needed

  • PBS: Phosphate buffered saline, pH 7.4
  • Fixation solution: 4% Paraformaldehyd in PBS, pH 7.4, 4% sucrose
  • Blocking buffer: 10% normal serum, 0.1% Triton X100 in PBS (normal serum from the host-species of the secondary antibodies is recommended for blocking)
  • Incubation buffer: 5% normal serum, 0.1% Triton X100 in PBS (Triton may be omitted; normal serum from the host-species of the secondary antibodies is recommended for blocking)
  • Mounting medium
  • Optional: DAPI nuclear stain

Procedure

  1. Wash cells briefly with PBS.
  2. Fix cells with fixation solution for 15 - 20 min at RT.
  3. Wash three times with PBS for 10 min.
  4. Incubate for 30 min with blocking buffer.
  5. Incubate in incubation solution containing the primary antibody (for appropriate dilution refer to the data sheet) for 2 h at RT.
  6. Wash three times with PBS for 10 min.
  7. Incubate in incubation solution containing the secondary antibody for 1 h at RT (optimal dilution must be determined experimentally). Optional: Add DAPI to the secondary antibody solution. Avoid bright light when working with the secondary antibody to minimize photo bleaching of the fluorescent dye. Important Note: This step can be omitted when fluorophore conjugated primary antibodies are used. In Multiplex staining, make sure to use secondary antibodies cross-adsorbed against the host species of the other primary antibody used in your experiment. Ideally, all secondary antibodies should come from the same host species. If not, make sure that they have been cross-adsorbed against IgGs of the host-species of the other secondary antibody as well. This avoids cross-reaction between the secondary antibodies.
  8. Wash three times with PBS for 10 min.
  9. Mount coverslips and observe under a microscope.

Note: The SYSY standard protocol generates good results in the SYSY labs and may be used as a reference. However, to achieve the highest specific signal and lowest non-specific background signal, the best antibody concentration, incubation temperature, and incubation time must be determined individually.