In Situ Immunofluorescence Analysis: Analyzing RNA Synthesis by 5-Bromouridine-5-Triphosphate Labeling

This technique is used to visualize sites of active transcription in a permeabilized cell and does not require radiolabeled molecules (e.g., [³ H] Uridine). Nonradioactive ribonucleic acid (RNA) precursors (e.g., 5-bromouridine-5′-triphosphate [BrUTP]) are used and can be detected by using fluorescently labeled antibodies. Procedures for BrUTP of labeling transcription sites require manipulations that are best applied to adherent cells but can be applied, with difficulty, to cell cultures in suspension. The protocol described below is for adherent cells grown on cover slips.