35 research outputs found
Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues
Abstract Paraffin sections from animal or human tissues fixed in different fixatives were submitted to immunostaining with the mouse monoclonal antibody 19A2, developed by Ogata et al. (1987a) against cyclin/PCNA. Detection of the bound antibody was performed by the indirect method with biotinylated sheep antibody and streptavidinâbiotinâperoxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, BouinâHollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanolâfixed tissues. Hydrolysis with 2 N HCl prior to immunocytochemistry (as currently performed to render incorporated BrdU accessible to antibodies) somewhat improved the results with Bouin or Carnoy and markedly augmented the intensity of the peroxidase reactions in formaldehyde and in methanolâfixed tissues. The distribution of the positive nuclei in the two latter cases coincided with the proliferative compartment. On the other hand, double labelling with [3H]âthymidine and with the cyclin/PCNA antibody revealed that in methanolâfixed tissues the cyclin/PCNA labelling index did not differ by more than 6% from the [3H]âthymidine index. Besides the two labels overlapped in a proportion of labelled cells that was in reasonable agreement with expectation considering cells flow in and out of S phase since the time of [3H]âthymidine injection. This indicates that both labels recognize the same cells in this material. In contrast, in formaldehydeâfixed tissues, the cyclin/PCNA labelling index markedly exceeded the [3H]âthymidine labelling index. From this it is concluded that cyclin/PCNA immunostaining can be used: âIn formaldehydeâfixed tissues (including existing material stored as paraffin blocks): for defining and mapping the proliferative (or germinative) compartment. âIn methanolâfixed tissues as a substitute to the [3H]âthymidine autoradiographic labelling index. From this, a method is proposed (derived from classical âdoubleâlabellingâtechnique) for measuring S phase duration in tissues fixed at a known interval time after a single labelling with [3H]âthymidine (or BrdU) and submitted to cyclin/PCNA immunocytochemical detection and to autoradiography (or to BrdU immunostaining). Copyright © 1989, Wiley Blackwell. All rights reservedSCOPUS: ar.jSCOPUS: ar.jinfo:eu-repo/semantics/publishe