Protocol

Analyzing DNA Replication II: Fixation and Processing of Tissues and Cells Labeled with Bromodeoxyuridine (BrdU)

This protocol was adapted from "Analyzing DNA Replication: Nonisotopic Labeling," Chapter 13, in Basic Methods in Microscopy, (eds. Spector and Goldman). Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, USA, 2006.

INTRODUCTION

The number of cells traversing the cell cycle and the rate of progression through it provide important indices of cell growth and tumorigenicity. S-phase cells can also be identified by their high content of DNA polymerase and proliferating cell nuclear antigen, a component of the leading-strand polymerase. Although both these markers can be detected rapidly and conveniently using the appropriate antibodies, neither are found exclusively in S-phase cells. Immunolabeling after incorporation of modified DNA precursors (e.g., 5-bromodeoxyuridine [BrdU, bromodeoxyuridine]) allows more rapid and precise detection of cells in S-phase of the cell cycle. BrdU is phosphorylated by cells to give BrdUTP, and this precursor is incorporated into DNA instead of deoxythimidine triphosphate. In living cells, BrdU is incorporated into replication sites that can then be detected using fluorochrome or enzyme-coupled antibodies. Alternatively, DNA synthesis sites can be labeled at high resolution by incubating cells with analogs of the natural precursors of DNA. The cells are then fixed and the incorporation sites are detected using fluorochrome- or enzyme-tagged antibodies. Formaldehyde- or alcohol-based fixation and paraffin embedding are used frequently. Formaldehyde, a mild protein cross-linking reagent, preserves cell structure well, and washing with a nonionic detergent or organic solvent permeabilizes membranes and allows antibodies access to the interior of the cell. This protocol describe the methods necessary to fix and process tissue and cell samples for subsequent antibody detection of loaded DNA precursors.