Immunofluorescence Troubleshooting Tips

Immunofluorescence (IF) technique combines the immunological method (specific binding of antigen and antibody) with fluorescence labeling method. The fluorescence produced by fluorescein could be detected with the fluorescence microscope, thus IF can be used in the localization analysis of specific antigens in cells.

Here Elabscience lists the common Immunofluorescence troubleshooting.

1. Weak fluorescence signals or no fluorescence expression

Possible causes 1：The target protein not present or low expressed in the sample

Suggestions 1：Use cells or tissues with high content of target protein

Possible causes 2：The antigen epitope was destroyed by immobilization step before staining

Suggestions 2：Choose another immobilization method

Possible causes 3：Poor cell permeability

Suggestions 3：Increase the concentration or reaction time of permeability agent

Possible causes 4：Antigen was lost due to the permeation

Suggestions 4：Decrease the concentration or reaction time of permeability agent

Possible causes 5：The primary/ secondary antibody concentration may be too low

Suggestions 5：Increase the primary/ secondary antibody concentration

Possible causes 6：Inappropriate secondary antibody

Suggestions 6：Ensure that the species of the secondary antibody matches the species of the primary antibody

1. High background of fluorescence

Possible causes 1：The primary antibody has a poor quality

Suggestions 1：Use primary antibody with good specificity and high titer

Possible causes 2：The primary/ secondary antibody concentration may be too high

Suggestions 2：Decrease the primary/ secondary antibody concentration

Possible causes 3：Insufficient blocking

Suggestions 3：Increase the blocking time

Possible causes 4：BSA of blocking buffer contains IgG

Suggestions 4：Use highly purified BSA (IgG free)

Possible causes 5：Insufficient washing

Suggestions 5：Increase the time and frequency of washing

Possible causes 6：The cell section has dried out

Suggestions 6：Keep the cell section at high humidity and do not let them dry out

Possible causes 7：Antigen was lost due to the permeation

Suggestions 7：Antigen was lost due to the permeation

Possible causes 8：The parameters of fluorescence microscope were not set correctly

Suggestions 8：Adjust the parameters of the fluorescence microscope to reduce background

1. Fast fluorescence quenching

Possible causes 1：The fluorescein has poor stability

Suggestions 1：Use fluorescein secondary antibody with good photo stability

Possible causes 2：The sealing agents which can prevent fluorescence from quenching has not been used

Suggestions 2：Use sealing agents to prevent fluorescence from quenching

1. Cell auto-fluorescence

Possible causes 1：No fluorescence quenching was performed after using glutaraldehyde as fixative

Suggestions 1：Detect the auto-fluorescence before staining, and operate the fluorescence quenching if there is auto-fluorescence

Possible causes 2：The sample itself (such as paraffin) has auto-fluorescence

Suggestions 2：Set negative control, and decrease the parameters of the fluorescence microscope to reduce background

Possible causes 3：The cell components (e.g. riboflavin, cytochrome, etc.) produce auto-fluorescence

Suggestions 3：Try to avoid using samples with high concentration of riboflavin and cytochrome and other cell components with auto-fluorescence

Possible causes 4：The ratio of dead cells/living cells is too high

Suggestions 4：Avoid cell death

Notes for fluorescent double staining

For Indirect method:

1) It is recommended to use primary antibodies from two different species, and secondary antibodies with different fluorescence labeling.

2) It is recommended to incubate one primary antibody, then incubate the fluorescence secondary antibody which matches the primary antibody, then followed with incubation of another primary antibody and its matched fluorescence secondary antibody.

3) Set positive control and negative control.

4) Use blocking serum that was collected from the species in which the secondary antibody was raised.

The other procedures are the same as conventional IF experiments.

For Direct method:

The species of primary antibodies are not specially required, and the fluorescent labels of two primary antibodies should be different.