Immunofluorescence microscopy
Summary
Immunofluorescence microscopy shows where in a cell a particular protein is located.
Also known as:
Immunofluorescence, IF
Samples needed
Fixed cells or tissue on a slide
Controls
Frequently, in addition to antibodies for the protein of interest, cells are also stained with DAPI. DAPI binds to DNA, thus marking the cell nucleus. When DNA-bound, DAPI fluoresces, emitting blue light.
Often times, other controls for IF are not shown in a published work. However, if a researcher is using new IF antibodies for the first time, several controls are useful. A positive control that contains the protein of interest tells the researcher if the antibody works well in IF and/or if the conditions used for staining are optimal. A control without the primary antibody ensures that the secondary antibody is binding specifically to the primary antibody and not other proteins. Finally, a control without the secondary antibody ensures that any observed signal is not autofluorescence of the cells or tissues.
Method
To stain a sample for immunofluorescence microscopy, samples are first fixed, then solubilized. Solubilizing creates holes in the plasma membrane to allow the antibodies to enter the sample. The sample is then blocked, often with milk protein, to discourage any non-specific binding of antibodies. Next, the sample is incubated in a primary antibody, which specifically binds to the protein of interest. Then, the sample is incubated in a secondary antibody, which binds to the primary antibody based on what species the primary antibody was raised in. (For instance, the secondary antibody might be something like anti-rabbit IgG.) The secondary antibody is chemically attached to a fluorescent molecule that allows detection with a fluorescence microscope.
Interpretation
Figure 1. Two immunofluorescence experiments assessing intracellular localization of protein RRP1B. Relevant section of caption for published figure reads: “A, co-IF followed by confocal microscopy was performed to define the cellular localization of RRP1B. Hemagglutinin-tagged, full-length RRP1B was ectopically expressed in HeLa cells to define its cellular localization. Cells were co-stained for ectopic RRP1B (red), either the nucleolar marker fibrillarin (FBL; top) or the nuclear envelope marker lamin B1 (LMNB1; bottom) (green), and double-stranded DNA (DAPI; blue). ” “Figure 2” by Nigel P.S. Crawford, Hailiu Yang, Katherine R. Mattaini, Kent W. Hunter[1]. [Image description]
The reader can tell that these images focus on cell nuclei, because of the blue rounded structures in the DAPI channel. The authors are interested in the protein RRP1B and want to determine where it is within the cell. In the top panels, cells have been stained for both RRP1B and the protein fibrillarin, which is known to localize to the nucleolus. The top right panel is the overlay of the red (RRP1B) and green (fibrillarin) channels. In the image, locations with both red and green sign