Tag Archives: FITC

About the header picture

This picture was taken by me a long time ago (6 years, i think). Contrary to the name of the blog, this is not GFP.

To take this picture, I performed Immunofluorescence experiment. The procedure is as follows:

Cells are treated with whatever you are invetigating (in this case – cells were treated with 0.5mM  arsenite for 30 min).

Cells are them fixed by a crosslinking agent (in this case, para-formaldehyde). As of this moment, the cells are dead, and macromolecules (proteins, DNA, RNA) are fixed in their place.

Cells are treated with detergent (to open pores in the membrane) and irrelevant protein (e.g. BSA) to block the possibility of non-specific binding of antibodies.

Cells are then incubated with an antibody that recognizes the protein of interest (primary antibody), and then with a secondary antibody that recognizes the primary antibody.

The secondary antibody is conjugated to a fluorofore – a small fluorescent molecule. In this case Fluorescein isothiocyanate (FITC), which is excited at 494nm and emits light at 518nm, i.e. green light.

This picture was taken by an epifluorescence microscope. You can see the cell morphology; you can detect the nucleus (large oval area in the middle); and you can also detect multiple granules in the cytoplasm (these are P-bodies and stress granules).  However, the picture isn’t sharp, for some cells it is out of focus, and in most cells, the fluorescent intensities of the nucleus and some granules glow too strong to allow good view of the nearby cytopalsm. Such problems can be resolved by using more advanced microscopy, like confocal. I hope that by next week I will have new, better pictures to show.