This method is used to visualise the localisation and quantity of a protein of interest. The target protein is bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore. A fluorescent or confocal microscope is used to visualise the protein. Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast, immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC, the sample requires permeabilisation so that the antibodies can reach the intracellular targets. Depending on the thickness of the sample, IHC samples do not require this. Do you have a technical question? Get in touch: info@stjohnslabs.com

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Protocol Series:
Immunocytochemistry
Information on the general steps required for carrying out Immunocytochemistry
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For more in our protocol series, visit Slideshare.net/StJohnsLabs

2. Immunocytochemistry is used to visualise the localisation and quantity of a protein of interest. The target protein is
bound to by a specific primary antibody, which in turn is detected by a secondary antibody conjugated to a fluorophore.
A fluorescent or confocal microscope is used to visualise the protein.
Immunocytochemistry (ICC) differs from immunohistochemistry (IHC) in that the former is performed on samples of
intact cells that have had most, if not all, of their surrounding extracellular matrix removed. In contrast,
immunohistochemical samples are sections of biological tissue, where each cell is surrounded by tissue architecture and
other cells normally found in the intact tissue. These differences cause the samples to be prepared differently. For ICC,
the sample requires permeabilisation so that the antibodies can reach the intracellular targets.
The following slides provide information on the general steps involved. Optimisation may be required.
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3. Cell Fixation
1. Apply 4% paraformaldehyde (PFA) to each well for 5 minutes or apply 100% methanol (chilled at -20°C) at
room temperature for 5 minutes.
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4. Blocking
1. Prepare the blocking solution by dissolving 0.5% Roche blocking reagent in 1x PBS and adding 5% donkey
serum.
2. Add the blocking solution to each well of the well-plate used for cell culture for 90 minutes at room
temperature.
3. Aspirate off the blocking solution.
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5. Antibody Staining
1. Dilute the primary antibody in blocking buffer using the recommended/optimised dilution. Incubate
overnight at 4°C.
2. Aspirate off the primary antibody.
3. Wash 4 times with 1x PBS (10 minutes per wash).
4. Dilute the secondary antibody in blocking buffer using the recommended/optimised dilution. Incubate for
1-3 hours at room temperature in the dark.
5. Aspirate off the secondary antibody.
6. Wash 4 times with 1x PBS (10 minutes per wash).
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6. Counterstaining
1. Counterstain the cell nuclei with 4,6-diamidino-2-phenylindole (DAPI) diluted 1:5000 in 1x PBS for 1 minute
at room temperature in the dark.
2. Wash with 1x PBS for 1 minute.
3. Wash with distilled water for 1 minute.
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7. Mounting
1. Mount the coverslips cell-side down onto microscope slides using mounting medium.
2. Seal with clear nail varnish and leave to dry.
3. Store at 4°C.
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8. Visualisation
1. View the coverslips under a fluorescent or confocal microscope to view the stained nuclei and target
protein.
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9. Download this protocol in PDF format:
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10. Antibody Validation Project
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