Cell Staining for Immunofluorescence Microscopy
1. FIXING THE CELLS
Coverslip Preparation for Adherent Cells

Grow cells on glass coverslips.
Rinse briefly in phosphate-buffered saline (PBS).
Coverslip Preparation for Non-Adherent Cells

Coat coverslips with an excess of poly-L-lysine (0.01% solution) for 10 minutes at room temperature.
Aspirate the poly-L-lysine solution and allow coverslips to dry completely.
Transfer cells in medium to 50ml tubes.
Centrifuge (400 x g, 15°C) for 5 minutes.
Aspirate the medium and resuspend the cells in 30ml phosphate-buffered saline (PBS).
Cover the dried, treated coverslips with the cell suspension.
Incubate at room temperature for 30–60 minutes.
Aspirate excess cell suspension.
Rinse briefly in PBS.
Formaldehyde Fixation

Immerse for 30 minutes in 3.7% formaldehyde solution at 37°C (for 100ml: 10ml 10X PBS, 33.4ml of 11.1% formaldehyde, 0.6ml of 30% Triton-X, 56ml distilled water). The solution is stable for 1 week at 4°C. Formaldehyde should be freshly prepared from its para-polymer. It is toxic and should be handled appropriately in a fume hood.

Methanol/Acetone Fixation

Immerse coverslips in ice-cold methanol:acetone (1:1) and incubate at -20°C for 10 minutes. Air dry the coverslips.

2. BLOCKING INCUBATION WITH PRIMARY ANTIBODY
Remove the blocking buffer by holding each coverslip on its edge with forceps and draining it onto a sheet of fiber-free paper.
Dilute primary antibody to 1.0–10 µg/ml in blocking buffer (optimal concentration will depend on several variables, such as the affinity of the antibody and the abundance of the antigen).
Distribute 50–100µl of the primary antibody solution on each coverslip and incubate for 1 hour at room temperature.
Decant the antibody solution or remove by aspiration.
Wash coverslips three times in PBS, 5 minutes each wash.
Incubation with more than one Primary Antibody

If it is desirable to examine the co-distribution of two different antigens in the same cell, a double immunofluorescence procedure may be used. Cells may be incubated simultaneously with two primary antibodies, provided they are monospecific and can be distinguished with secondary antibodies conjugated to different fluorochromes (or with primary antibodies directly conjugated to different fluorochromes).

Incubate coverslips with the mixture of diluted primary antibodies for 1 hour at room temperature.
Decant the antibody solution or remove by aspiration.
Wash coverslips three times in PBS, 5 minutes each wash.

3. INCUBATION WITH SECONDARY ANTIBODIES
Note: If the primary antibodies are already conjugated to a fluorochrome, incubation with secondary antibody is unnecessary.

The coverslips are now incubated with secondary antibodies conjugated to a fluorochrome; e.g. anti-mouse IgG:FITC or Cy3, depending on the donor species of the primary antibody and the desired fluorochrome. We recommend the use of cross-adsorbed and affinity-purified secondary antibodies to minimize background and non-specific reactivity from the secondary antibody. High-quality conjugated antibodies are essential for the avoidance of cross reactivity between two different antibodies in double immunofluorescence protocols.

Place coverslips cells-side-up on a piece of filter paper inside a petri dish.
Dilute the secondary antibodies to the appropriate concentration in blocking buffer. Add enough secondary antibody solution to cover the surface of each coverslip (usually 50–100µl).
Incubate for 1 hour at room temperature.
Remove the secondary antibody by blotting the edge of each coverslip on fiber-free paper.
Wash coverslips three times in PBS, 5 minutes each wash.

4. PREPARATION FOR MICROSCOPY
Invert each coverslip onto a slide containing 10µl of mounting media.
Remove the excess mounting media with fiber-free paper, without disturbing the coverslip. Seal the edges of each coverslip with regular transparent nail polish and allow to dry for 3 minutes. This will provide semi-permanent preparations. The cells are now ready for microscopic viewing.