Multicolor immunostaining (not really FISH, but...)
(Multicolor) fluorescent immunostainingguidlines
Slide preparation, cell fixation, permeabilization and antibody incubations are performed similar to the "FISH + immunostaining" page, but omitting the denaturing and hybridization.
For multicolor immunostaining, as for multicolor FISH, one has to pay special attention to potential cross-reactivity among antibodies from different species. Therefore, antibodies should be added in a "logical" step-wise approach, to prevent these nteractions from taking place. The use of colors depends on the filter availability in your fluorescence microscope. With a good filter set, up to eight different fluorophores can be simultaneously discriminated on the same preparation. A more thorough discussion on fluorophores and colors is presented in the multicolor FISH, CCK and nucleotide labeling sections.
A few examples of immunostaining are shown in the figure below.All nuclei were stained blue (DAPI). The two images on the left show the distribution of the actin fibers in wild-type and mutant fibroblasts (mutation for a gene involved in phoshphorylation). The figure at the bottom right corner, shows the distribution of vimentin (part of the desmosomes) at the junctions between cells. The large image shows part of a fibroblasts in which the actin fibers were detected in red, whereas vinculin (part of the focal adhesions) was detected green.
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Last modified on: Feb12, 2001