Ultrastructural cryoimmunocytochemistry is a convenient tool for the study of DNA replication in cultured cells.
In the present study, we have optimized an immunocytochemical ultrastructural approach for in situ localization of newly synthesized DNA in unsynchronized as well as in synchronized human HeLa cells and in exponentially growing mouse P815 cells, which had incorporated bromodeoxyuridine (BrdU) during short pulses varying from 1 to 20 minutes. The incorporated BrdU was detected in hydrolyzed ultrathin cryosections or Lowicryl sections by means of a monoclonal antibody, revealed by secondary colloidal gold-labeled probes. The results demonstrate our ability to study, with high resolution and reproducibility, DNA replication during consecutive periods of the S-phase, which is monitored by the incorporation of tritiated thymidine. In addition, this approach allows one to perform a concomitant mapping of replicated DNA and various enzymes of the replisome.
Raska
I
I
Laboratory of Ultrastructural Analysis, University of Lausanne, Switzerland.
Michel
L S
LS
Jarnik
M
M
Dundr
M
M
Fakan
S
S
Gasser
S
S
Gassmann
M
M
Hübscher
U
U
Izaurralde
E
E
Martinez
E
E
D000818:Animals; D000906:Antibodies; D002453:Cell Cycle / Q000187:drug effects; D004247:DNA / Q000648:ultrastructure*; D004261:DNA Replication; D005615:Freezing; D006367:HeLa Cells; D006801:Humans; D007150:Immunohistochemistry / Q000379:methods*; D051379:Mice; D016253:Microscopy, Immunoelectron / Q000379:methods*; D013936:Thymidine / Q000494:pharmacology; D014407:Tumor Cells, Cultured
D000906:Antibodies; D004247:DNA; D013936:Thymidine