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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.
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.