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BD Biosciences Support - Protocols - Cell Bio - Cell Staining for Immunofluorescence Microscopy

Support Protocols

Cell Staining for Immunofluorescence Microscopy


Coverslip Preparation for Adherent Cells

  1. Grow cells on glass coverslips or glass chamber slides.
  2. Rinse briefly in phosphate-buffered saline (PBS).

Coverslip Preparation for Non-Adherent Cells

  1. Coat coverslips with an excess of poly-L-lysine (0.01% solution, Sigma, cat.# P4707) for 10 minutes at room temperature.
  2. Aspirate the poly-L-lysine solution and allow coverslips to dry completely.
  3. Transfer cells in medium to 50 ml tubes.
  4. Centrifuge (400 x g, 15°C) for 5 minutes.
  5. Aspirate the medium and resuspend the cells in 30 ml phosphate-buffered saline (PBS).
  6. Cover the dried, treated coverslips with the cell suspension.
  7. Incubate at room temperature for 30–60 minutes.
  8. Aspirate excess cell suspension.
  9. Rinse briefly in PBS.

Paraformaldehyde Fixation

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

Methanol/Acetone Fixation

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


Place the coverslips cells-side-up in a petri dish. Rinse the cells with PBS, then cover the cells with blocking buffer (1% BSA in PBS) for 30 minutes at 37°C to minimize non-specific adsorption of the antibodies to the coverslip (150-200 µl is usually sufficient to completely cover the surface area).


  1. Remove the blocking buffer by holding each coverslip on its edge with forceps and draining it onto a sheet of fiber-free paper.
  2. 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).
  3. Distribute 150-200 µl of the primary antibody solution on each coverslip or chamber and incubate for 1 hour at room temperature in a humidified chamber.
  4. Decant the antibody solution or remove by aspiration.
  5. Wash coverslips or chamber slides three times in PBS, 5 minutes each wash.


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).

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


Note: If the primary antibodies are already conjugated to a fluorochrome, incubation with secondary antibody is not necessary.

The coverslips are 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.

  1. Place coverslips cells-side-up in a petri dish.
  2. Dilute the secondary antibody to the appropriate concentration in blocking buffer. Add enough secondary antibody solution to cover the surface of each coverslip (usually 150-200 µl) or add secondary antibody to each chamber of the chamber slides.
  3. Incubate for 1 hour at room temperature.
  4. Remove the secondary antibody by blotting the edge of each coverslip on fiber-free paper.
  5. Wash coverslips three times in PBS, 5 minutes each wash.


  1. Invert each coverslip onto a slide containing 10 µl of mounting media (VECTASHIELD mounting media; Vector Laboratories cat. #H-1000) or detach the chamber wells from the glass slide and add mounting media.
  2. 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.