Fluorescence in situ hybridization of cells on polyvinylidene fluoride membrane filters.

Figure 2A shows an example of a whole-mount embryo double-labeled with a fluorescein-labeled fushi tarazu (ftz) and a digoxigenin labeled wingless (wg) probe. Several other combinations of differentially labeled probes were tested including a fluorescein-labeled ftzprobe together with a digoxigenin-labeled engrailed(en) probe, a fluores-cein-labeled wgprobe together with a digoxigenin-labeled en probe and a flu-orescein-labeled wg probe together with a digoxigenin-labeled wgprobe. All combinations worked equally well (data not shown). We also tested whether this type of FISH were compatible with immuno-fluorescence-based protein detection. Figure 2B shows an embryo that was labeled for wgmRNA (red) and en protein (green). The mRNA probe preparation, hybridization and post-hy-bridization washes were carried out as described for the double in situ protocol above, except that a shorter proteinase K treatment (half the time) was used to reduce degradation of protein epitopes. In this experiment, a digoxigenin-labeled wg probe was used because the protein being visualized was detected using a mouse anti-Engrailed mono-clonal antibody (Antibody 4D9; Reference 5). After the post-hybridization washes and final rinse in PBTB, embryos were incubated with anti-digoxi-genin antibody as described above and the anti-En antibody (ascites diluted 1:500). Note that the combination of these two approaches at this point saves considerable time (about 24 h) over previously published nonfluorescence double-labeling techniques (4) and avoids deterioration of the first signal (usually the product of alkaline phos-phatase staining) during detection of the second. Subsequent washes in PBTB were performed as described above followed by incubations with the appropriate secondary antibodies (donkey anti-sheep CY3 and goat anti-mouse CY2). After final washing, embryos were mounted and observed as described above in the double in situ protocol. A FISH/antibody double-labeling protocol for Drosophilaembryos was also recently described by Goto and Hayashi (1). Their protocol makes use of the alkaline phosphatase substrate Fast Red TR for fluorescence detection of digoxigenin-labeled RNA probes. We haven't adequately tested this approach to see how it compares in terms of sensitivity and convenience. However , the diffusible nature of the alkaline phosphatase product, as opposed to fluorescently conjugated antibodies, would likely lower resolution significantly at the subcellular level. Cell migration within the embryonic limb primordium of Drosophilaas revealed by a novel fluores-cence method to visualize mRNA and protein. A non-radioactive in situ hybridization method for the localization of specific RNAs in Drosophila embryos reveals translational control of the segmentation gene hunchback. Chromosoma 98 :81-85. Many cells are lost during sample preparation …