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Direct staining refers to the use of a labeled primary antibody. Direct staining facilitates multiplexing as it allows using primary antibodies generated in the same species.
Indirect staining corresponds to the use of labeled secondary antibodies and is based on the pairing of a primary with a secondary antibody. Usually, the secondary antibody is specific for the host species of the primary antibody limiting the multiplexing application. Indirect staining is generally preferred for low abundance target antigen as it demonstrates higher sensitivity compared to direct staining as several secondary antibodies can bind a single primary antibody.
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As every scientific experiment, immunofluorescence requires controls to make sure that your antibody is correctly detecting its target and not to observe false-positive results.
Here are some of the controls that are regularly undertaken in immunofluorescence.
Positive controls can be obtained by testing a cell line known to express your protein of interest.
Commonly used negative controls include:
Tests on cell lines known not to express the protein of interest such as KO cell lines or cells with gene mediated silencing.
No primary control: in the “no primary control”, the assays conditions remain exactly the same as the “normal” experimental conditions except that the primary antibody is not added. The goal of this control is to test the specificity of the primary antibody.
Isotype control: the isotype control is commonly used to determine the background level of the sample. The isotype control necessitates an antibody of the same isotype, clonality, label and host species of the primary antibody but should detect a molecule not present in the sample. The experimental conditions should be identical to those used with the primary antibody.
Immunofluorescence is based on the use of an antibody-label conjugate to determine the localization and expression of target proteins in fixed cells or tissues. The antibody-label conjugates combine two properties which make them particularly suitable for microscopy experiments:
The specificity of the antibody allowing to detect only the targeted antigen
The “detectability” of the label
There are mainly two types of immunofluorescence:
Direct immunofluorescence which is based on the use of a primary antibody conjugated to a fluorophore
Indirect immunofluorescence which is based on the use of a labeled secondary antibody recognizing the primary.
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