DNA synthesis in excised tobacco leaves after bromodeoxyuridine incorporation: immunohistochemical detection in semi-thin Spurr sections

Abstract

We describe a method for localizing replicating cells in detached tobacco leaves allowed to root. The proposed protocol has shown that formalin fixation and Spurr embedding of petiole bases can be used for demonstrating DNA synthesis after bromodeoxyuridine (BrdU) incorporation. The incorporated BrdU was immunologically visualized. After resin removal, different procedures of DNA denaturation and protease digestion were tested. Combined hydrolysis with 4 N HCl for 10 min at room temperature and digestion with 0.4% pepsin for 15 min at 37°C led to the best reproducible results, with either the peroxidase or the gold detection system. The method is rapid and sensitive, with precise resolution. It can be used at the light and electron microscopic levels, Its potential application is to elucidate in the same organ the role of cytokinins, a class of plant growth regulations, in dividing cells and to define the chronology of their biosynthesis in roots in relation to DNA synthesis.