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IF Staining Controls

Learn about the controls to include in your IF experiments.

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IF Staining Controls

In order to understand and interpret the IF images obtained, different control stainings can be performed that help to reveal unspecific staining. 

  • The unstained sample (just fixed, blocked, permeabilized) should be analyzed to understand the autofluorescence background signal. 

  • A sample (just fixed, blocked, permeabilized) that is incubated with the secondary antibody reveals whether the secondary antibody binding is specific. 

  • To ensure specific binding of the primary antibody, the sample can be blocked with a specific blocking peptide (used to raise the primary antibody). Binding of the primary antibody will be inhibited.

  • In case of multistainings, the staining should also be performed separately to ensure no cross-reactions and appropriate labeling.

 

Potential IF drawbacks

As mentioned at the beginning of this guide, IF staining is a powerful tool with many benefits when used in/for analysis of a target of interest. Conversely, there are certain drawbacks about which you should be aware:

  • The fixation step results in the killing of cells and dynamic and fast processes cannot be monitored. An IF image is a snapshot of the cell at a certain point in time.

  • Fixation or permeabilization also result in artifacts. In order to ensure that no artifacts are observed, it is necessary to perform a couple of additional time-consuming controls. 

  • The storage time for IF samples is short due to photobleaching and the limited stability of the fluorescent label.