Thermo Fisher Scientific is committed to antibody performance and specificity testing. To support this commitment, each Invitrogen antibody that is indicated for immunofluorescence applications has been tested using a protocol similar to that provided below. These tests help confirm our antibody’s performance and help ensure superior results when used in your experiments.

The typical immunofluorescence protocol (for both adherent and suspension cells) that Invitrogen antibodies are subjected to is reproduced below. This protocol includes:

Immunofluorescence for adherent cells

Immunofluorescence for suspension cells

Note: Some of the steps in this protocol require optimization depending on the sample and antibody being used.

Immunofluorescence protocol for adherent cells

Cell preparation for adherent cells

  1. Seed 1–1.5 x104 cells per well of a 4-chamber slide in 500 mL of culture medium. Incubate at 37°C at 5% CO2.
  2. 32–36 hours post cell seeding, remove the cell culture medium and rinse the cells 3 times using 500 µL of 1X PBS.

Fixation

Paraformaldehyde as fixative

  1. Fix cells using 400 µL of 4% paraformaldehyde (pH 7.4) for 10 min at 37°C.
  2. Remove paraformaldehyde solution and wash the cells 3 times with 500 µL of 1X PBS.

Methanol as fixative

  1. Fix cells using 400 µL of 100% ice-cold methanol for 5 min at –20°C.
  2. Remove methanol and wash the cells three times with 500 µL of 1X PBS.

Permeabilization

  1. Add 400 µL of 0.1% Triton X-100 in 1X PBS and incubate the cells at room temperature for 15 min.
  2. Remove Triton-X-100 and wash the cells three times with 500 µL of 1X PBS.

Blocking

Add 500 µL of 2% BSA in 1X PBS and incubate the cells at room temperature for 60 min. (Alternately, the cells can be incubated overnight in 2% BSA or 1X PBS before proceeding to immunostaining.)

Immunostaining

  1. Add the desired concentration of primary antibody diluted in 500 µL of 0.1% BSA to the cells and incubate for 3 hours at room temperature or overnight at 4°C.
  2. Remove primary antibody solution and wash the cells three times with 500 µL of 1X PBS.
  3. Add the desired concentration of fluorescent dye–labeled secondary antibody along with a compatible counterstain for the cytoskeleton (e.g., rhodamine phalloidin) and nucleus (DAPI) diluted in 500 µL of 0.1% BSA and incubate for 45 min at room temperature protected from light.
  4. Wash the cells three times with 500 µL of 1X PBS-T.

Note: Use extra wells for controls.

  • Control #1—without antibodies, only include counterstains.
  • Control #2—with fluorescent dye–labeled secondary antibody only, without including primary antibody to test for specificity of fluorescent staining and to avoid artifacts based on autofluorescence of the cells.

    Note: Optimize concentration of counterstains, primary and secondary antibody dilutions, as well as fixation, blocking and washing steps for best results.   

Mounting

  1. Air-dry the coverslip/chamber slide and add the mounting medium (containing antifade agent).
  2. If desired, seal the coverslips and visualize the target antigen by fluorescence microscopy.

Immunofluorescence protocol for suspension cells

Cell preparation for suspension cells

  1. Centrifuge the cell suspension at 1,500 rpm for 5 min, discard supernatant.
  2. Wash cells with 1 mL of 1X PBS, and obtain a pellet by centrifugation at 1,500 rpm for 5 min.

Fixation

Paraformaldehyde as fixative

  1. Add 700 µL of 4% paraformaldehyde (pH 7.4) to the cell pellet, mix thoroughly and incubate for 10 min at 37°C followed by centrifugation at 1,500 rpm for 5 min.
  2. Discard the supernatant, add 800 µL of 1X PBS to the pellet, and mix thoroughly. Centrifuge at 1,500 rpm for 5 min.

Methanol as fixative

  1. Incubate the cells with 700 µL 100% ice-cold methanol for 5 min at –20°C followed by centrifugation at 1,500 rpm for 5 min.
  2. Discard supernatant and mix thoroughly with 800 µL of 1X PBS and centrifuge at 1,500 rpm for 5 min.

Permeabilization

  1. Add 700 µL of 0.1% Triton X-100 in 1X PBS and incubate the cells at room temperature for 15 min followed by pelleting at 1,500 rpm for 5 min.
  2. Discard the supernatant and add 800 µL of 1X PBS to the pellet, mix thoroughly and centrifuge at 1,500 rpm for 5 min.

Blocking

Add 1 mL 2% BSA in 1X PBS for 3.5 million cells. Incubate the cells at room temperature for 60 min. (Alternately, the cells can be incubated overnight in 2% BSA or 1X PBS before proceeding to immunostaining.)

Immunostaining

  1. Add the desired concentration of primary antibody diluted in 500 µL of 0.1% BSA to the cells and incubate for 3 hours at room temperature or overnight at 4°C.
  2. Remove primary antibody solution and wash the cells three times with 500 µL of 1X PBS.
  3. Add the desired concentration of fluorescent dye–labeled secondary antibody along with a compatible counterstain for the cytoskeleton (e.g., rhodamine phalloidin) and nucleus (DAPI) diluted in 500 µL of 0.1% BSA and incubate for 45 min at room temperature protected from light.
  4. Wash the cells three times with 500 µL of 1X PBS-T.

Note: Use extra wells for controls.

  • Control #1—without antibodies, only include counterstains.
  • Control #2—with fluorescent dye–labeled secondary antibody only, without including primary antibody to test for specificity of fluorescent staining and to avoid artifacts based on autofluorescence of the cells.

    Note: Optimize concentration of counterstains, primary and secondary antibody dilutions, as well as fixation, blocking and washing steps for best results.  

Mounting

  1. Air-dry the coverslip/chamber slide and add the mounting medium (containing antifade agent).
  2. If desired, seal the coverslips and visualize the target antigen by fluorescence microscopy.

NOTE

For more detailed protocols and additional tips and tricks, go to the Molecular Probes School of Fluorescence.

For Research Use Only. Not for use in diagnostic procedures.