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BrdU labeling of HeLa cells followed by immunostaining
Fluorescence microscopy for cell suspensions
In an immunofluorescence (IF) experiment a primary antibody binds specifically to a protein of interest present in a sample (e.g. fixed cells, tissue sections). The antibody binding is then visualized by a fluorescent detection system (hence the name immunofluorescence), which provides information about the localization of the protein. Two types of IF staining methods exist:
In the indirect staining protocol, several secondary/tertiary antibodies carrying multiple labels bind to the primary antibody, resulting in signal amplification. In the protocol for direct staining, using a directly conjugated antibody, there is no signal amplification step. This might result in weak staining or no staining at all if the target protein is present at low levels. Therefore, the use of directly conjugated antibodies in IF is only recommended for the detection of very abundant target proteins.
Recently, multi-color IF experiments that simultaneously use several different fluorophore have increased in popularity. When designing multi-color IF experiments it is crucial to follow a few simple rules: