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There are a number of factors that influence how easily an antigen may be detected. How specific is the antibody and how diffuse is the antigen? The ideal is a large number of binding sites in a small area; but if the antigen is diffuse enough, it may be difficult to detect it from a high background signal.
The type of fixative used and its subsequent method of application are in large part empirically determined. Many fixatives alter the architectural integrity of the cell and the
antibody may no longer recognize its epitope. Using frozen sections causes minimal alteration of the antigen but at the cost of poor preservation of cellular morphology. Sensitivity may further be influenced by the detection method used. Fluorochrome-labeled antibodies are excellent for visualizing subcellular components at high magnification and resolution, while enzyme-labeled antibodies offer higher sensitivity.
Polyclonal, monoclonal, and pooled monoclonal antibody preparations are generally used. Polyclonal serum will yield a strong signal, but requires careful titration to avoid an unacceptably high background. This background can be lowered by preabsorbing the serum with a saturating amount of competitor protein, either in the form of nonspecific proteins provided by BSA or serum from the antibody host.
The purity and specificity of monoclonal antibodies results in a low background over a wide spectrum of concentrations. Generally, monoclonal antibodies work well with cells fixed in organic solvents or paraformaldehyde. The monoclonal antibodies that are used should be from tissue culture supernatants or purified from tissue culture supernatants or ascites fluid. Using whole ascites fluid, a not uncommon source for monoclonal antibodies, will yield unacceptably high levels of contaminating antibodies. Further amplification of the signal may be obtained by using a biotin-avidin amplification system. In these instances, one molecule (e.g., biotin) is bound to the antibody, which is bound to the antigen.
Then, several molecules (e.g., avidin) linked to the detection molecule (e.g., fluorescein) bind to each biotin, thus amplifying the signal.
Cells to be used for fixation and staining can be grown on coverslips, in chamber slides, or tissue culture dishes. If chamber slides are used, they should be glass slides and should be coated with a matrix to which the cells will adhere. This is also true of the glass coverslips. Plastic tissue-culture-treated slides and coverslips can be used if the cells are not to be used with a fluorescent antibody. If glass coverslips are used, they should be rinsed and stored in acetonexethanol. They can then be flame dried or rinsed in PBS or medium just prior to use. Suspension cultures can be grown in the usual manner and centrifuged onto slides using a centrifuge like the "cytospin" designed for this purpose.
Cell Preparation, Fixation, and Antibody Binding
1. Chamber slides, 8-well, or coverslips
2. Fibronectin (Bovine, Sigma, catalogue No. F1411)
3. Growth medium
4. 4% paraformaldehyde solution
5. 0.1 M glycine
7. 1% Triton-X in PBS
8. 3% BSA in PBS
9. Tris-buffered saline
11. Tris buffer Procedure
1. Trypsinize and neutralize the trypsin in the cell suspension.
2. Seed the chambers with a cell density that will give good growth to 50% confluency within 48"C72 hr. The idea is to avoid a tightly packed, confluent chamber.
3. When the cells are growing well but only semiconfluent, remove the medium and wash 1 x with serum free F12"CDME.
4. Mix an equal volume of F12"CDME and 4% paraformaldehyde (for a final concentration of 2%). Add to each chamber well and leave for 20 min at room temperature.
5. Wash 1x with PBS and add 0.1 M glycine for 20 min.
6. Wash with PBS 2x.
7. Permeabilize with 1% Triton-x 100 in PBS for 6 min. at room temperature.
8. Rinse 2x in PBS (leave a coating a detergent with each wash).
9. Remove all but 200^l PBS. Apply the first antibody, 200 ^l/well (1:25 if dilution is unknown).
10. Incubate for 30 hr at 37jaC.
11. Wash 4x in PBS plus 0.1% Tween-20 (5 min./rinse). Leave 200 ^l in well each rinse.
12. Add the secondary antibody in 3% BSA"CPBS or 10% host serum.
13. Repeat steps 9"C11.
14. If using an alkaline phosphatase detection method, wash in Tris buffer for 5 min.
The detection step uses either enzyme-labeled or fluorescent-labeled reagents. The enzyme label is detected using a chromogenic substrate that precipitates subsequent to the enzyme reaction. This results in an insoluble colored product at the site of localization of the antigen. Secondary antibodies conjugated to either horseradish peroxidase or alkaline phosphatase are commercially available, and when used with a wide range of substrates can produce several different colored products that vary in intensity. The use of a variety of substrates may be required, depending on the level of background and sensitivity, and when two colors are required. For horseradish peroxidase, the most common substrate is diaminobenzidine (DAB), which yields an intense dark bronze or brown color. If DAB yields too high a background, chloronaphthol (blue-black) or aminoethylcarbazole (AEC), which gives a less intense red color, can be used. For alkaline phosphatase, the most common chromagen is bromochloroindolyl phosphate"Cnitro blue tetrazolium (BCIP"CNBT), which yields a purple-black color. Naphthol-AS-Bl-phosphate"Cnew fuchsin (NABP"CNF) or fast red is also frequently used. These colored products produce a permanent record and the slides can be stored and reviewed after long periods of time, whereas fluorescent probes fade with time and some are readily quenched during the process of viewing the slides.