In situ fluorescence visualization of bromouridine incorporated into newlytranscribed nucleolar RNA

Bromouridine-triphosphate is commonly used for in situ immunocytochemical l abeling of newly synthesized RNA in living cells. While extranucleolar tran scripts do not require special conditions for visualization, special treatm ent prior to fixation (e. g. incubation with alpha-amanitine) is necessary for immunofluorescence detection of bromouridine-labeled nucleolar RNA in p revious studies. We show in the present investigation that bromouridine-tri phosphate is efficiently used by both extranucleolar and nucleolar RNA poly merases in living cultured cells. The failure to detect incorporated bromou ridine within nucleoli is entirely due to improper treatment of cells after bromouridine incorporation. When methanol/acetone fixation is used, fluore scence signals within nucleoli can be routinely found.