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Page
designed and maintained by Octavian Henegariu
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WARNING:
The information provided in these pages is
copyrighted and is intended for individual use
only. No parts of this work (text, tables or
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Multicolor immunostaining (not really
FISH, but...)
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Slide preparation, cell
fixation, permeabilization and antibody incubations are performed
similar to the "FISH + immunostaining" page, but omitting
the denaturing and hybridization.
- After placing the cells on slides (for example using a cytospin
or simply by growing them on slides or coverslips), they are fixed
in methanol or ethanol, either at room temperature for 1 hour, or
by heating them 1 minute at 65 C in ethanol (chemical
aging).
- Cell permeabilization can be done by 1-5 minute incubation in a
solution containing almost any detergent (SDS, saponin, Tween 20,
NP-40) at concentrations of 0.05%-0.2% (lower for SDS, higher for
the other !!) in PBS or other isotonic buffer.
- All antibodies were diluted in 4xSSC at 1:100 to 1:500
dilutions of 0.5-1 mg/ml stocks. All antibody incubations were
done 10 minutes at 37 C.
- Between any primary and secondary antibody incubation, the slide
is rinsed three times 5 minutes each, in a jar with 4xSSC or with
4xSSC/0.1% Tween20 (which decreases somewhat the background, and does
not affect binding of the IgG antibody)
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.
