Immunocytochemistry protocol

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Immunocytochemistry protocol

Immunocytochemistry (ICC) is a widely used technique to study the expression situation of specific protein or antigen in cells. The target molecule is recognized and combined by specific primary antibody. The primary antibody is bound by a secondary antibody labeled with fluorophore so that it allows visualization of the target molecule under a fluorescence microscope. ICC helps researchers to know whether or not the target molecule is expressed in cells and which subcellular compartment the target molecule exists.

Fig. The schematic diagram for immunocytochemistry.

Immunocytochemistry protocol


Primary antibody
Secondary antibody
Serum (depends on the host of the secondary antibody)
Hoechst 33258 or DAPI

Cell culture

  1. For immunocytochemistry, cells are grown on coverslips and cells should not reach over 80% confluency as recommendation.

Cell fixation

  1. Remove the medium and wash cells once with PBS (pH 7.4).
  2. Cells are fixed with 4% p -formaldehyde in PBS for 20 min at room temperature.
  3. Wash cells twice with PBS (pH 7.4).
  4. Incubate cells with 0.1% Triton-X100 for 5 min Permeabilize cell membrane.
  5. Wash cells twice with PBS (pH 7.4).


  1. Block nonspecific binding sites in PBS for 1 h with 10% serum which depends on the host of the secondary antibody.
  2. Primary antibodies (1-2 ug/ml) and secondary antibodies (5-10 ug/ml) are diluted in 0.1% serum in PBS. Incubate cell with primary antibody at 37 °C for 2 h or at 4 °C overnight.
  3. Wash with PBS 3 times for 15 min each.
  4. Incubate cell with secondary antibody at 37 °C for 1 h protecting from light.
  5. Wash with PBS 3 times for 15 min each.
  6. Nuclei are stained with Hoechst 33258 or DAPI in PBS for 15 min at room temperature.
  7. Coverslips are then mounted with medium for fluorescent staining.
  8. Once the medium dried, the slides are imaged with confocal fluorescence microscopy.

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