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
- Place a previously autoclaved, 13 mm circular glass coverslip in each well of a 24-well plate.
- Plate approximately 1 mL of cell suspension into each well. Incubate 24 hours in a 37°C CO2 incubator.
- Aspirate media from wells.
- Add appropriate fixative and incubate for the appropriate time at room temperature.
- Wash the cells with PBS, twice, for 15 minutes. Do not shake.
- Cover cells with 400 µL of 1% BSA in PBS and incubate for 1 hour at room temperature.
- Wash the cells with PBS for 15 minutes.
- Incubate the cells with primary antibody of interest in 1% BSA in PBS and incubate for 2 hours at room temperature.
- Wash the cells twice with PBS for 5 minutes.
- 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.
- Wash the cells three times with PBS.
- Aspirate well dry.
- Clean a glass slide with Alconox® and water, follow with a rinse in 70% ETOH. Dry using a Kimwipe®.
- Place a drop of Aqua Poly-Mount® mounting media on cleaned slide.
- 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.
- Let dry at room temperature, seal edge if desired and examine the cells under a fluorescent microscope.
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