Immunofluorescence Labeling of Cells

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.

Reagents and Equipment

1. Cultured cells grown on coverslips
2. PBS: 0.01M Phosphate buffered saline, pH 7.2-7.4 (P3813 or P4417)
3.  
   1. Methanol, cooled at -20 °C for at least 1 hr (Product No. 32213)
   2. Acetone, cooled at -20 °C for at least 1 hr (Product No. 24201)

   or

   1. 3-4% Paraformaldehyde (Product No. P6148) in PBS. Dissolve in PBS, using 5N NaOH and mild heating
   2. 0.5% Triton X-100 (Product No. T9284)  in PBS

   or

   1. 3-4% Paraformaldehyde (Product No. P6148) in PBS. Dissolve in PBS, using 5N NaOH and mild heating
   2. Methanol, cooled at -20 °C for at least 1 hr (Product No. 32213.)

   or

   1. PEM Buffer: 0.1M PIPES (Product No. P8203), 5 mM EGTA (Product No. E4378), 2mM MgCl₂ · 6H₂O (Product No. M0250). Bring to pH 6.8, using NaOH solution
   2. Ethanol, cooled at -20 °C for at least 1 hr (Product No. 270741)
4. Primary antibody
5. Antibody controls
6. Secondary antibody-fluorochrome-labeled
7. Aqueous mounting medium
8. Microscope glass slides (76 x 25 mm. Product No. S8902)
9. Fluorescence microscope, equipped with appropriate filter for fluorescence detection
10. Inverted light microscope (e.g. Nikon TMS)

Procedure

Cell Preparation
Fixation
Application of Primary Antibody
Application of Secondary Antibody
Evaluation

Notes:

1. This is provided as a general protocol. Optimal dilutions for the primary and secondary antibodies, cells preparation, controls, as well as incubation times will need to be determined empirically and may require extensive titration. Ideally, one would use the primary antibody as recommended in product data sheet. The appropriate negative and positive controls should also be included.
2. Refer to the corresponding SDS for safe handling of chemicals required.
3. Protect fluorescent conjugates and labeled slides from the light. Incubate samples in the dark and cover whenever possible.

Cell Preparation

1. Remove cells from incubator. Inspect under inverted light microscope to verify the desired appearance. Discard medium.
2. Rinse with PBS; remove excess solution.

Fixation

Fix cells:

Four different fixation methods are described. Choose the appropriate fixation method according to the application (or product data sheet recommendation).

Methanol-Acetone Fixation

1. Fix in cooled methanol, 10 minutes at –20 °C.
2. Remove excess methanol.
3. Permeabilize with cooled acetone for 1 minute at –20 °C.

or
Paraformaldehyde-Triton Fixation

1. Fix in 3-4% paraformaldehyde for 10-20 minutes.
2. Rinse briefly with PBS.
3. Permeabilize with 0.5% Triton X-100 for 2-10 minutes.

or
Paraformaldehyde-Methanol Fixation

1. Fix in 3-4% paraformaldehyde for 10-20 minutes.
2. Rinse briefly with PBS.
3. Permeabilize with cooled methanol for 5-10 minutes at –20 °C.

or
PEM-Ethanol Fixation

1. Fix in PEM buffer for 10 minutes.
2. Rinse twice, briefly, with PBS.
3. Permeabilize with cooled ethanol for 5-10 minutes at –20 °C.

Wash three times (at least 5 minutes each) with PBS.

Application of Primary Antibody

1. Dilute primary antibody in PBS to appropriate dilution. Apply on coverslips over the cells and incubate for 60 minutes at room temperature (it is recommended to use a humidified chamber).
2. Wash three times (at least 5 minutes each) with PBS.

Application of Secondary Antibody

Note: Secondary antibody is applied only in indirect assays.

1. Dilute labeled secondary antibody to appropriate dilution in PBS. Apply on coverslips and incubate for 30 minutes at room temperature.
2. Wash three times (at least 5 minutes each) with PBS.
3. Remove excess PBS.

Evaluation

1. Mount coverslips with mounting medium and invert onto glass slides.
2. Inspect under the microscope.
3. Record the results. It is recommended to photograph the labeled cells.

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.

References

1. Giloh H, Sedat J. 1982. Fluorescence microscopy: reduced photobleaching of rhodamine and fluorescein protein conjugates by n-propyl gallate. Science. 217(4566):1252-1255. https://doi.org/10.1126/science.7112126

2. Storz H, Jelke E. 1984. Photomicrography of weakly fluorescent objects ? employment of p-phenylene diamine as a blocker of fading. Acta Histochemica. 75(2):133-139. https://doi.org/10.1016/s0065-1281(84)80048-3

3. Harlow E, Lane D. 1988. Antibodies: A Laboratory Manual. 1. New York: Cold Spring Harbor Larboratory Press.

4. Johnson G, Davidson R, McNamee K, Russell G, Goodwin D, Holborow E. 1982. Fading of immunofluorescence during microscopy: a study of the phenomenon and its remedy. Journal of Immunological Methods. 55(2):231-242. https://doi.org/10.1016/0022-1759(82)90035-7

5. Longin A, Souchier C, Ffrench M, Bryon PA. 1993. Comparison of anti-fading agents used in fluorescence microscopy: image analysis and laser confocal microscopy study.. J Histochem Cytochem.. 41(12):1833-1840. https://doi.org/10.1177/41.12.8245431

6. Beesley J. 1993. Immunocytochemistry: A Practical Approach. Oxford University Press.

7. Bullock G, Petrusz P. 1989. Techniques in Immunocytochemistry. 1. Academic Press.