Immunocytochemistry General Protocol

Immunocytochemistry (ICC) by definition is the demonstration of a tissue constituent in situ by detecting specific antibody-antigen interactions where the antibody has been tagged with a visible label. The visual marker may be a fluorescent dye, colloidal metal, hapten, radioactive marker or more commonly, an enzyme for light microscopy. Experimental samples ranging from frozen sections, cell culture/suspension, to whole tissue samples have been used. Ideally, maximal signal strength along with minimal background or non-specific staining are required to give optimal antigen demonstration.

It is recommended to review the methodology and variations in protocols from: Immunocytochemical Methods and Protocols (second edition), edited by Lorette C. Javois, from Methods in Molecular Medicine, Humana Press, 1999 (ISBN 0-89603-570-0).

Protocol

1. Place a previously autoclaved, 13 mm circular glass coverslip in each well of a 24-well plate.
2. Plate approximately 1 mL of cell suspension into each well. Incubate 24 hours in a 37°C CO2 incubator.
3. Aspirate media from wells.
4. Add appropriate fixative and incubate for the appropriate time at room temperature.
5. Wash the cells with PBS, twice, for 15 minutes. Do not shake.
6. Cover cells with 400 µL of 1% BSA in PBS and incubate for 1 hour at room temperature.
7. Wash the cells with PBS for 15 minutes.
8. Incubate the cells with primary antibody of interest in 1% BSA in PBS and incubate for 2 hours at room temperature.
9. Wash the cells twice with PBS for 5 minutes.
10. Incubate the cells with a dilution of IgG fluorescein conjugated secondary antibody of choice in 1% BSA in PBS for 1 hour at room temperature.
11. Wash the cells three times with PBS.
12. Aspirate well dry.
13. Clean a glass slide with Alconox® and water, follow with a rinse in 70% ETOH. Dry using a Kimwipe®.
14. Place a drop of Aqua Poly-Mount® mounting media on cleaned slide.
15. Using forceps and/or a 26 gauge needle with the tip bent, retrieve the glass coverslip from the well and place it cell side down on top of the drop of mounting media.
16. Let dry at room temperature, seal edge if desired and examine the cells under a fluorescent microscope.

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