Protocol of Immunofluorescence

The protocols described below are for general application. Any product specific protocol supercedes these general recommendations. The typical immunofluorescence protocols include:

• Protocol A: Protocol of Immunofluorescence for Adherent Cells
• Protocol B: Protocol of Immunofluorescence for Suspension Cells

General Steps:

1. Cell preparation
2. Fixation
3. Permeabilization
4. Blocking
5. Immunostaining
6. Mounting

Refer to our Troubleshooting of Immunofluorescence when you are in trouble. If necessary, please don’t hesitate to contact us for additional help.

Protocol A: Protocol of Immunofluorescence for Adherent Cells

Reagents

• 1X PBS
• 4% Paraformaldehyde (pH 7.4)
• Optional: Methanol
• 2% BSA in 1X PBS
• 0.1% BSA in 1X PBS
• 1X PBS-T
• Primary Antibody
• Fluorochrome-conjugated Secondary Antibody

Methods

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 h post cell seeding, remove the cell culture medium and rinse the cells three times using 500 µL 1X PBS.
3. Paraformaldehyde as fixative: Fix cells using 400 µL 4% paraformaldehyde for 10 min at 37°C. Remove paraformaldehyde solution. Wash the cells three times with 500 µL 1X PBS.
(Optional for Methanol as fixative: Fix cells using 400 µL totally ice-cold methanol for 5 min at -20°C. Remove methanol. Wash the cells three times with 500 µL 1X PBS.)
4. Add 500 µL 2% BSA. Incubate the cells at RT for 1 h. Alternately, the cells can be incubated overnight in 2% BSA or 1X PBS before proceeding to immunostaining.
5. Add the desired concentration of primary antibody diluted in 500 µL 0.1% BSA to the cells. Incubate for 3 h at RT or overnight at 4°C.
6. Remove primary antibody solution. Wash the cells three times with 500 µL 1X PBS.
7. 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 0.1% BSA and incubate for 45 min at RT. Avoid light.
8. Wash the cells three times with 500 µL 1X PBS-T.
9. Air-dry the coverslip/chamber slide and add the mounting medium (containing antifade agent). If desired, seal the coverslips and visualize the target antigen by fluorescence microscopy.

Protocol B: Protocol of Immunofluorescence for Suspension Cells

Reagents

• 1X PBS
• 4% Paraformaldehyde (pH 7.4)
• Optional: Methanol
• 0.1% Triton X-100 in 1X PBS
• 2% BSA in 1X PBS
• 0.1% BSA in 1X PBS
• 1X PBS-T
• Primary Antibody
• Fluorochrome-conjugated Secondary Antibody

Methods

1. Centrifuge the cell suspension at 1,500 rpm for 5 min. Discard supernatant.
2. Wash cells with 1 mL 1X PBS. Centrifuge at 1,500 rpm for 5 min to obtain a pellet.
3. Paraformaldehyde as fixative: Add 700 µL 4% paraformaldehyde to the cell pellet. Mix thoroughly. Incubate for 10 min at 37°C. Centrifuge at 1,500 rpm for 5 min. Discard supernatant. Add 800 µL 1X PBS to the pellet. Mix thoroughly. Centrifuge at 1,500 rpm for 5 min.
(Optional for Methanol as fixative: Incubate the cells with 700 µL totally ice-cold methanol for 5 min at -20°C. Centrifuge at 1,500 rpm for 5 min. Discard supernatant. Mix thoroughly with 800 µL 1X PBS and centrifuge at 1,500 rpm for 5 min.)
4. Add 700 µL 0.1% Triton X-100 in 1X PBS. Incubate the cells at RT for 15 min followed by pelleting at 1,500 rpm for 5 min.
5. Discard supernatant. Add 800 µL 1X PBS to the pellet. Mix thoroughly. Centrifuge at 1,500 rpm for 5 min.
6. Add 1 mL 2% BSA for 3.5 million cells. Incubate the cells at RT for 1 h. Alternately, the cells can be incubated overnight in 2% BSA or 1X PBS before proceeding to immunostaining.
7. Add the desired concentration of primary antibody diluted in 500 µL 0.1% BSA to the cells. Incubate for 3 h at RT or overnight at 4°C.
8. Remove primary antibody solution. Wash the cells three times with 500 µL 1X PBS.
9. 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 0.1% BSA. Incubate for 45 min at RT. Avoid light.
10. Wash the cells three times with 500 µL 1X PBS-T.
11. Air-dry the coverslip/chamber slide and add the mounting medium (containing antifade agent). If desired, seal the coverslips and visualize the target antigen by fluorescence microscopy.

Notes

1. Some of the steps in this protocol require optimization depending on the sample and antibody being used.
2. Optimize concentration of counterstains, primary and secondary antibody dilutions, as well as fixation, blocking and washing steps for best results.
3. Extra wells are needed for controls. Control 1-without antibodies, only include counterstains. Control 2-with fluorescent dye-labeled secondary antibody only.