S phase duration measurement by combined PCNA/cyclin immunostaining and radioautography after a single pulse-labelling with 3H-thymidine.
A method for measuring S phase duration is described and evaluated that combines single pulse labelling with 3H-thymidine (TdR), detected by radioautography, and proliferating cell nuclear antigen (PCNA)/cyclin immunostaining to replace the second pulse labelling of the classical double-labelling method. Conditions were set up in which nuclei showing one or both types of label were readily distinguished, hence allowing to verify that cell fluxes in and out of S phase were equal. S phase durations thus measured in different tissues of the mouse were concordant with those obtained by the double 3H-TdR labelling or from labelled mitoses curves. Our method might be used with archived samples of methanol-fixed cells or tissues, singly labelled with 3H-TdR or with bromodeoxyuridine.
Morret-Rauis
M
M
Department of Internal Medicine and Laboratory of Hematology, Hopital Universitaire Brugmann, ULB, Brussels, Belgium.
Degraef
C
C
Galand
P
P
D000818:Animals; D016213:Cyclins / Q000032:analysis; D003584:Cytological Techniques; D004261:DNA Replication; D005260:Female; D005836:Genitalia, Female / Q000166:cytology; D007422:Intestines / Q000166:cytology; D051379:Mice; D009687:Nuclear Proteins / Q000032:analysis; D018809:Proliferating Cell Nuclear Antigen; D016196:S Phase; D013936:Thymidine; D013997:Time Factors
D016213:Cyclins; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D013936:Thymidine