The sensitivity and resolution of the in situ hybridization procedure has been enhanced with the use of fluorescent dendrimers as probes for mRNA. Dendrimers revealed the presence of MyoD mRNA two days earlier than previously detected with enzymatic or radiolabeled probes (2
). Single cells containing a low level of MyoD mRNA were resolved due to the preciseness of the fluorescent signal and the minimal level of background from nonspecific binding.
Messenger RNA can be detected at low levels in whole embryos at early stages of development and in tissue sections. Dendrimers also can be used to localize mRNA in cultured cells. The ability to label dendrimers with a variety of fluorochromes permits the localization of different mRNAs in the same tissue section. Dendrimers also can be tagged with 32
P, biotin, and digoxigenin. Furthermore, in situ hybridizations with dendrimers can be combined with antibody labeling to localize mRNA and protein within the same cell. This capability led to the discovery of a cell surface antibody for cells expressing MyoD mRNA that was then used to isolate MyoD mRNA positive cells from the embryo and fetal organs (9
). The specificity of MyoD dendrimers for cells with myogenic potential was confirmed by demonstrating that the isolated population differentiated into skeletal muscle in culture (9
The protocol for using dendrimers is straightforward, particularly for those experienced with conventional methods of in situ hybridization. The usual considerations of avoiding RNAase and DNAase contamination, selecting anti-sense oligonucleotide sequences that remain linear, and optimization of hybridization temperature also apply when using dendrimer probes. A unique feature of the dendrimer procedure is the use of pepsin instead of Proteinase K. The appropriate temperature and length of incubation with pepsin must be carefully determined. Exposure to pepsin for 10 minutes at room temperature results in adequate digestion of most tissues for dendrimer penetration and preserves morphology. However, dense tissues, such as liver, require a 12-minute treatment with pepsin at room temperature, while the more fragile tissues of the early embryo are treated for only 5 minutes at 4°C.
Enzymatically labeled oligonucleotide probes that are smaller than dendrimers are particularly useful when performing whole mount in situ hybridization with older embryos and thicker tissues. While enzymatic probes are capable of amplifying the signal, resolution can be compromised due to the diffusion of the reaction product and accumulation of background. Neither dendrimers nor enzymatically labeled oligonucleotides are quantitative in tissue sections; however, relative comparisons of signal intensity can be made with both types of probes. Although the sensitivity of dendrimers needs to be determined, conceivably, a single mRNA could be detected with dendrimers due to the number of fluors present on each molecule.
The most significant advantages of using fluorescent dendrimers for in situ hybridization are their low level of background from nonspecific binding and pinpoint emission from the fluorescent tag. A single labeled cell can be detected among thousands of non-expressing cells. Observing the “needle in the haystack” is not only critical for determining the onset of gene expression in a subpopulation of cells within the embryo, but for detecting stem cells in adult tissues and early metastasis of cancer cells.