Antibodies are an important tool for demonstrating both the presence and the subcellular localization of an antigen. Cell staining is a very versatile technique and, if the antigen is highly localized, can detect as few as a thousand antigen molecules in a cell. In some circumstances, cell staining may also be used to determine the approximate concentration of an antigen, especially by an image analyzer. Cell staining can be divided into four steps: cell preparation, fixation, application of antibody, and evaluation
As a first step, cells to be stained are attached to a solid support to allow easy handling in subsequent procedures. This can be achieved by several methods: adherent cells may be grown on microscope slides, coverslips, or an optically suitable plastic support. Suspension cells can be centrifuged onto glass slides, bound to solid support using chemical linkers, or in some cases handled in suspension.
The second step is to fix and permeabilize the cells, to ensure free access of the antibody to its antigen. Perfect fixation would immobilize the antigens, while retaining authentic cellular and subcellular architecture and permitting unhindered access of antibodies to all cells and subcellular compartments. Wide ranges of fixatives are commonly used, and the correct choice of method will depend on the nature of the antigen being examined and on the properties of the antibody used. Fixation methods fall generally into two classes: 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 reduce the antigenicity of some cell components, and require the addition of a permeabilization step, to allow access of the antibody to the specimen. Fixation with both methods may denature protein antigens, and for this reason, antibodies prepared against denatured proteins may be more useful for cell staining. Four different fixation methods are described. The appropriate fixation method should be chosen according to the relevant application.
The third step of cell staining involves incubation of cell preparations with antibody. Unbound antibody is removed by washing, and the bound antibody is detected either directly (if the primary antibody is labeled) or, indirectly using a fluorochrome-labeled secondary reagent.
In the fourth and final step, the staining is evaluated using fluorescence microscopy.
Four different fixation methods are described. Choose the appropriate fixation method according to the application (or product data sheet recommendation).
Wash three times (at least 5 minutes each) with PBS.
Note: Secondary antibody is applied only in indirect assays.
Note: It is advisable to run the appropriate negative controls. Negative controls establish background fluorescence and non-specific staining of the primary and secondary antibodies. The ideal negative control reagent is a fluorochrome conjugated mouse monoclonal or myeloma protein. It should be isotype-matched, not specific for cells of the species being studied and of the same concentration as the test antibody. The degree of autofluorescence or negative control reagent fluorescence will vary with the type of cells under study and the sensitivity of the instrument used. For fluorescent analysis of cells with Fc receptors, the use of isotype-matched negative controls is mandatory.