Protocol of Multicolor Immunofluorescence (IF)

The protocols described below are for general application. Any product specific protocol supercedes these general recommendations. The typical IF protocols include the following steps:

Step 1: Cell Culture

Step 2: Fixation

Step 3: Permeabilization

Step 4: Blocking

Step 5: Multicolor Immunostaining

• Protocol A: Simultaneous Incubation with Directly Labeled Primary Antibodies (Direct Detection)
• Protocol B: Simultaneous Incubation with Unlabeled Primary Antibodies (Indirect Detection)
• Protocol C: Sequential Incubations with Unlabeled Antibodies (Indirect Detection)

Step 6: Counterstaining, Mounting and Imaging

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

Reagents:

• Poly-L-lysine
• PBS
• PBS-T
• Blocking buffer: 5% BSA in PBS-T
• 4% Paraformaldehyde

Optional: Methanol

• 0.5% Triton X-100 in PBS
• Normal serum
• Antibodies

Methods

Step 1: Cell Culture

• Coat the coverslips with poly-L-lysine. Dry and sterilize them.
• Seed the cells on poly-L-lysine coated sterile glass coverslips. Grow the cells to semi-confluency.

(Some cell types may require growth on other treated coverslips for proper adhesion.)

Step 2: Fixation

1. Aspirate the culture medium from the dish or remove each coverslip as required with tweezers. Gently wash them with PBS at RT.
2. Incubate the coverslips in freshly prepared 4% paraformaldehyde for 10 minutes. Alternatively, the cells can be fixed for 10 minutes in methanol (pre-equilibrated at -20°C) on ice. Wash the coverslips in PBS for 2 minutes.
(Alternative fixation methods may be tested and compared to determine which is best at preserving the structure and epitope of the protein of interest.)

Step 3: Permeabilization

1. Incubate the coverslips in 0.5% Triton X-100 in PBS at RT for 5 minutes. Test different detergents in a range of concentrations to find the optimal condition. Wash the coverslips in PBS for 5 minutes.
(Step 3 is not required with methanol fixation.)

Step 4: Blocking

1. IgG from the secondary antibody may bind non-specifically to the sticky sites on the cells which often leads to non-specific background signal. To avoid this issue, block the coverslips in 1-5% normal serum prepared in PBS for one hour at RT.
(The normal serum block should be of the same species in which the secondary antibody has been raised. For instance, if you are using a goat-anti rabbit secondary antibody, then block with 5% normal goat serum. For Protocol B, the blocking buffer can be prepared by mixing the serum from each host of the secondary antibodies.)

Step 5: Multicolor Immunostaining

Multicolor labeling experiments are best carried out by sequentially incubating cells with primary and secondary antibodies. It may be performed by using one of the following three protocols.

Protocol A: Simultaneous Incubation with Directly Labeled Primary Antibodies (Direct Detection)

1. After Step 4, incubate the cells with directly labeled primary antibodies in the blocking buffer in a humidified chamber for 1 h at RT or overnight at 4°C in the dark.
2. Decant the antibody solution. Wash the cells three times in PBS-T for 5 minutes for each wash.

Note:

• This protocol is useful when the primary antibodies are from the same host. For example, a mouse monoclonal antibody (mAb) against antigen-1 and a mouse mAb against antigen-2.

Protocol B: Simultaneous Incubation with Unlabeled Primary Antibodies (Indirect Detection)

1. After Step 4, incubate the cells with unlabeled primary antibodies in the blocking buffer in a humidified chamber for 1 h at RT or overnight at 4°C.
2. Decant the antibody solution. Wash the cells three times in PBS-T for 5 minutes for each wash.
3. Incubate the cells with both secondary antibodies in 1% BSA for 1 h at RT in the dark.
4. Decant the secondary antibody solution. Wash the cells three times in PBS-T for 5 minutes for each wash in the dark.

Note:

• This method is useful when the primary antibodies are from different hosts. For example, a mouse mAb against antigen-1 and rabbit polyclonal antibody (pAb) against antigen-2.

Protocol C: Sequential Incubations with Unlabeled Antibodies (Indirect Detection)

1. Blocking Step #1:

Incubate cells with blocking buffer solution #1 for 30 minutes at RT. This blocking buffer solution contains 5% serum from the same species which is the same as the host of the labeled secondary antibody #1.

2. Primary Antibody Incubation #1:

Incubate cells with the primary antibody #1 in 1% BSA or 1% serum in a humidified chamber for 1 h at RT or overnight at 4°C.

3. Decant the primary antibody solution. Wash the cells three times in PBS-T for 5 minutes for each wash.
4. Secondary Antibody Incubation #1:

Incubate cells with secondary antibody #1 in 1% BSA for 1 h at RT in the dark.

5. Decant the secondary antibody solution. Wash the cells three times in PBS-T for 5 minutes for each wash.
6. Blocking Step #2:

Incubate cells with the blocking buffer #2 for 30 minutes at RT in the dark. This blocking buffer solution contains 5% serum from the same species which is the same as the host of the labeled secondary antibody #2.

7. Primary Antibody Incubation #2:

Incubate cells with primary antibody #2 in 1% BSA or 1% serum in a humidified chamber for 1 h at RT or overnight at 4°C.

8. Decant the primary antibody solution. Wash the cells three times in PBS-T for 5 minutes for each wash.
9. Secondary Antibody Incubation #2:

Incubate cells with secondary antibody #2 in 1% BSA for 1 h at RT in the dark.

10. Decant the secondary antibody solution. Wash the cells three times in PBS-T for 5 minutes for each wash.

Note:

• This method is useful when the primary antibodies are from different hosts (a mouse mAb against antigen-1, rabbit pAb against antigen-2 and sheep pAb against antigen-3), and the antibodies display aggregates formation in the simultaneous incubation method.

Step 6: Counterstaining, Mounting and Imaging

1. When all the washing steps have been completed, cell nuclei can be counterstained with DAPI or Hoechst (1-10 µg/mL).
2. Invert the coverslip onto a glass slip with a drop of mounting media containing a fluorescence anti-fade agent.
3. Carefully remove the excess mounting media.
4. Examine the cells under a fluorescence microscope and image as required.