The use of 33P-labeled oligonucleotides for in situ hybridization of vertebrate embryo frozen sections.

In situ hybridization was performed on frozen sections of whole E16 rat embryos or whole E7 chick embryos using oligonucleotide probes tagged with a 33P-poly A tail. We used two antisense oligonucleotide probes: a 57-mer rat agrin oligo and a 60-mer chicken B-cadherin oligo. After exposure to Hyperfilm beta max for three days, mRNA was detected in distinct organs and tissues of the embryo. After dipping the slides in liquid emulsion and exposing them for two weeks, the grains could be localized to a specific cellular layer, for example, the ganglion layer of the retina or the luminal epithelium of the gizzard. It is concluded that 33P autoradiography is excellent for defining gross areas or cellular layers of mRNA in a vertebrate embryo with a resolution of about 20 microns.