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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:
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:
There are mainly two types of immunofluorescence:
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