Immunofluorescence Troubleshooting Tips

2018-05-28Author:adminpraise:1Download

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

Suggestions

  The target protein not present or low expressed in the sample

  Use cells or tissues with high content of target protein

  The antigen epitope was destroyed by immobilization step before staining

  Choose another immobilization method

  Poor cell permeability

  Increase the concentration or reaction time of permeability agent

  Antigen was lost due to the permeation

  Decrease the concentration or reaction time of permeability agent

  The primary/ secondary antibody concentration may be too low

  Increase the primary/ secondary antibody concentration

  Inappropriate secondary antibody

  Ensure that the species of the secondary antibody matches the species of the primary antibody


2.    High background of fluorescence

Possible causes

Suggestions

  The primary antibody has a poor quality

  Use primary antibody with good specificity and high titer

  The primary/ secondary antibody concentration may be too high

  Decrease the primary/ secondary antibody concentration

  Insufficient blocking

  Increase the blocking time

  BSA of blocking buffer contains IgG

  Use highly purified BSA (IgG free)

  Insufficient washing

  Increase the time and frequency of washing

  The cell section has dried out

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

  Antigen was lost due to the permeation

  Decrease the concentration or reaction time of permeability agent

  The parameters of fluorescence microscope were not set correctly

  Adjust the parameters of the fluorescence microscope to reduce background


3.    Fast fluorescence quenching

Possible causes

Suggestions

  The fluorescein has poor stability

  Use fluorescein secondary antibody with good photo stability

  The sealing agents which can prevent fluorescence from quenching has not been used

  Use sealing agents to prevent fluorescence from quenching


4.    Cell auto-fluorescence

Possible causes

Suggestions

No fluorescence quenching was performed after using glutaraldehyde as fixative

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

The sample itself (such as paraffin) has auto-fluorescence

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

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

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

The ratio of dead cells/living cells is too high 

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.