Fig. 2.

Retention of pol II transcriptional activity by transcription loops in oil-isolated nuclei. a Pol II immunostaining of lampbrush loops in a fixed spread prepared from an oocyte nucleus that had been isolated into oil and kept for about 3 h prior to spread preparation. The α-pol II monoclonal antibody that stains the loop axes recognizes a CTD phosphoisomer associated with transcriptionally active pol II. The brightly immunostained objects are B snurposomes, which contain epitopes that also cross-react with this antibody (Doyle et al. 2002). b Continued loop RNA synthesis in oil-isolated nuclei detected by Br-U incorporation followed by immunostaining. As summarized in the diagram, nuclei were isolated into oil and pre-incubated for about 2 h prior to injection of Br-UTP. Following incubation in oil for a further 3 h, each nucleus was transferred into a drop of oil under an aqueous solution into which it was then pushed, allowing the production of a fixed nuclear spread preparation. Immunostaining with an α-BrU antibody demonstrates that RNA synthesis was occurring on lampbrush loops at least 2 h after nuclear isolation (left-hand panels). As controls, a nuclear spread was prepared directly from an oocyte that had been injected 3 h previously with Br-UTP (center panels) and from an oil-isolated nucleus that was not injected with Br-UTP prior to incubation (right panels). Images are reproduced using the same contrast function