Immunofluorescence

Products, protocols, tips & tricks for all your IF needs

BrdU labeling of HeLa cells followed by immunostaining

BrdU labeling of HeLa cells followed by immunostaining

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:

  • Direct staining, which uses primary antibodies conjugated to fluorophore
  • Indirect staining, which mainly uses secondary antibodies labeled to fluorophore

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.

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Tips and tricks

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: 

  1. Select bright fluorophore that are highly photostable and have a low susceptibility to photobleaching. Conventional fluorophore such as FITC and R-PE are especially prone to photobleaching, which results in quick fading of the fluorescence signal. To reduce photobleaching use next generation fluorophore with high photostability, such as Alexa Fluor® or DyLight® Fluor dyes. Alternatively, photobleaching can be reduced by using mounting media with antifade reagents.
  2. If the emission spectra of two fluorophore overlap, one fluorophore might spill/bleed into another fluorophore filter. Bleed-through can make it difficult or sometimes impossible to distinguish the two fluorophore. When selecting fluorophore the emission and excitation spectra should therefore be checked for spectral overlap with the help of a fluorescence spectrum viewer. Ideally, there should be no overlap between the fluorophore used in your IF experiment.
  3. After having selected the fluorophore for your experiment you have to decide which antigen to detect with which fluorophore. The brightest fluorophore should be reserved for the detection of the antigen with the least abundant expression level. The dimmest fluorophore should be used for detecting the most abundant antigen.