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• Immunocytochemistry

Immunocytochemistry

The protocols described below are for general application. Any product specific protocol supercedes these general recommendations.

Background

Immunocytochemistry is an immunological technique that is very similar to Immunohistochemistry. Immunocytochemistry is used to visualize the presence of a specific protein or antigen in cells (cultured cells, cell suspensions), rather than tissues. The types of cell samples that can be investigated include blood smears, cultured cells, cell suspensions, and cytospins. Each type of cell sample is prepared and treated slightly differently, but the fundamental use of primary antibody, secondary antibody and color development is very similar between Immunocytochemistry and Immunohistochemistry.

For immunocytochemistry, sample preparation involves fixing the target cells to a slide. Cells can be attached to a solid surface by several methods: adherent cells may be grown on microscope slides; cell suspensions can be centrifuged onto glass slides (cytospin),or bound to solid support using chemical linkers.

To ensure access of the antibody to its antigen, cells must be fixed and permeabilized. In an ideal situation, fixation would immobilize the antigens while retaining native cellular architecture and permitting unhindered access of antibodies to all cells and subcellular compartments.

Fixation methods fall generally into two groups: organic solvents and cross-linking reagents. Organic solvents, such as alcohols and acetone, remove lipids and dehydrate the cells, while precipitating the proteins on the cellular architecture. Cross-linking reagents (such as paraformaldehyde) form intermolecular bridges, normally through free amino groups, thus creating a network of linked antigens. Cross-linkers preserve cell structure better than organic solvents, but may alter the structure of some cell components, so much so that they are not recognized by the primary antibody.

Fixation Methods

1. Acetone Fixation Fix cells in -20°C acetone for 5-10 minutes. No permeabilization step is needed following acetone fixation.
2. Methanol Fixation Fix cells in -20°C methanol for 5-10 minutes. No permeabilization step needed following methanol fixation.
3. Ethanol Fixation Fix cells in cooled 95% ethanol, 5% glacial acetic acid for 5-10 minutes.
4. Methanol-Acetone Fixation Fix cells in cooled methanol, 10 minutes at -20°C. Remove excess methanol. Permeabilize with cooled acetone for 1 minute at –20°C.
5. Methanol-Acetone Fixation Prepare 1:1 methanol and acetone mixture and fix cells at -20°C for 5-10 minutes.
6. Methanol-Ethanol Fixation Prepare 1:1 methanol and ethanol mixture and fix cells at -20°C for 5-10 minutes.
7. Formalin Fixation Fix cells in 10% neutral buffered formalin for 5-10 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.
8. Paraformaldehyde-Triton Fixation Fix cells in 3-4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with 0.5% Triton X-100 for 10 minutes.
9. Paraformaldehyde-Methanol Fixation Fix cells in 4% paraformaldehyde for 10-20 minutes. Rinse briefly with PBS. Permeabilize with cooled methanol for 5-10 minutes at -20°C.

Permeabilization Methods

Cross-linkers typically require the addition of a permeabilization step to allow access of the antibody to the specimen. Two groups of compounds can be used for permeabilization: organic solvents and mild surfactants. Alcohols and acetone can be used as a one step fixation/permeabilization method. In addition to their fixative behavior which precipitates proteins in place, these solvents also dissolve membrane lipids which render the membrane permeable to antibodies. In the surfactant category, saponin, Triton X-100, Tween-20, etc, are used for their ability to disrupt membranes.