DOI: 10.1111/j.1365-2184.1989.tb00223.x. Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues

Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues

10.1111/j.1365-2184.1989.tb00223.x

Crossref journal-article

Wiley

Cell Proliferation (311)

Abstract

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 streptavidin‐biotin‐peroxidase complexes. No, or faint, nuclear staining was seen in material fixed in ethanol, Bouin, Bouin‐Hollande, Carnoy or formaldehyde, whereas readily detectable immunocytochemical reaction was constantly observed over nuclei of methanol‐fixed 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 methanol‐fixed 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]‐thymidine and with the cyclin/PCNA antibody revealed that in methanol‐fixed tissues the cyclin/PCNA labelling index did not differ by more than 6% from the [3H]‐thymidine 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]‐thymidine injection. This indicates that both labels recognize the same cells in this material. In contrast, in formaldehyde‐fixed tissues, the cyclin/PCNA labelling index markedly exceeded the [3H]‐thymidine labelling index. From this it is concluded that cyclin/PCNA immunostaining can be used: In formaldehyde‐fixed tissues (including existing material stored as paraffin blocks): for defining and mapping the proliferative (or germinative) compartment. In methanol‐fixed tissues as a substitute to the [3H]‐thymidine autoradiographic labelling index. From this, a method is proposed (derived from classical ‘double‐labelling’technique) for measuring S phase duration in tissues fixed at a known interval time after a single labelling with [3H]‐thymidine (or BrdU) and submitted to cyclin/PCNA immunocytochemical detection and to autoradiography (or to BrdU immunostaining).

Bibliography

Galand, P., & Degraef, C. (1989). Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues. Cell Proliferation, 22(5), 383â392. Portico.

Authors 2

P. Galand (first)
C. Degraef (additional)

References 24 Referenced 5

10.1038/bjc.1977.234
{'key': 'e_1_2_1_3_1', 'first-page': '1291', 'article-title': 'Proliferating cell nuclear antigen expression is a reliable marker of the proliferative compartment (abstract)', 'volume': '92', 'author': 'Ahnen D.J.', 'year': '1987', 'journal-title': 'Gastroenterology'} / Gastroenterology / Proliferating cell nuclear antigen expression is a reliable marker of the proliferative compartment (abstract) by Ahnen D.J. (1987)
{'key': 'e_1_2_1_4_1', 'first-page': '325', 'article-title': 'In vitro autoradiographic determination of cell kinetic parameters in adenocarcinomas and adjacent healthy mucosa of the human colon and rectum', 'volume': '36', 'author': 'Bleiberg H.', 'year': '1976', 'journal-title': 'Cancer Res.'} / Cancer Res. / In vitro autoradiographic determination of cell kinetic parameters in adenocarcinomas and adjacent healthy mucosa of the human colon and rectum by Bleiberg H. (1976)
10.1083/jcb.84.3.795
10.1016/0014-4827(81)90009-4
10.1038/326515a0
10.1002/j.1460-2075.1985.tb03679.x
10.1083/jcb.105.4.1549
10.1002/j.1460-2075.1985.tb03758.x
{'key': 'e_1_2_1_11_1', 'first-page': '167', 'article-title': "Comparaison de deux méthodes autoradiographiques basées sur l'emploi de thymidine tritiée par la mesure de la durée de la phase S (phase de synthèse d'aide désoxyribonucléique) et de l'interphase des cellules de differents tissus de la souris", 'volume': '78', 'author': 'Galand P.', 'year': '1967', 'journal-title': 'Arch. Biol. (Liège)'} / Arch. Biol. (Liège) / Comparaison de deux méthodes autoradiographiques basées sur l'emploi de thymidine tritiée par la mesure de la durée de la phase S (phase de synthèse d'aide désoxyribonucléique) et de l'interphase des cellules de differents tissus de la souris by Galand P. (1967)
{'key': 'e_1_2_1_12_1', 'first-page': '37', 'article-title': 'In vitro autoradiographic studies of cell proliferation in the gastrointestinal tract of man', 'volume': '9', 'author': 'Galand P.', 'year': '1968', 'journal-title': 'J. Nuclear Med.'} / J. Nuclear Med. / In vitro autoradiographic studies of cell proliferation in the gastrointestinal tract of man by Galand P. (1968)
{'key': 'e_1_2_1_13_1', 'first-page': '733', 'article-title': 'Analysis of proliferative grade using anti PCNA/cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis', 'volume': '134', 'author': 'Garcia R.L.', 'year': '1989', 'journal-title': 'Am. J. Pathol.'} / Am. J. Pathol. / Analysis of proliferative grade using anti PCNA/cyclin monoclonal antibodies in fixed, embedded tissues. Comparison with flow cytometric analysis by Garcia R.L. (1989)
10.1016/0014-4827(86)90520-3
10.1038/309374a0
10.4049/jimmunol.121.6.2228
10.1177/33.8.3860561
10.1038/326542a0
10.1016/0014-4827(87)90020-6
10.4049/jimmunol.139.9.2942
10.1038/326517a0
10.1177/35.11.3116075
10.1007/BF00494803
10.1084/jem.154.6.1899
10.1007/BF00224731

Dates

Type	When
Created	17 years, 3 months ago (May 1, 2008, 5:47 p.m.)
Deposited	1 year, 10 months ago (Oct. 22, 2023, 4:35 p.m.)
Indexed	1 year, 2 months ago (June 17, 2024, 3:18 p.m.)
Issued	35 years, 11 months ago (Sept. 1, 1989)
Published	35 years, 11 months ago (Sept. 1, 1989)
Published Online	17 years, 3 months ago (May 1, 2008)
Published Print	35 years, 11 months ago (Sept. 1, 1989)

Funders 0

None

BibTeX

@article{Galand_1989, title={Cyclin/PCNA immunostaining as an alternative to tritiated thymidine pulse labelling for marking S phase cells in paraffin sections from animal and human tissues}, volume={22}, ISSN={1365-2184}, url={http://dx.doi.org/10.1111/j.1365-2184.1989.tb00223.x}, DOI={10.1111/j.1365-2184.1989.tb00223.x}, number={5}, journal={Cell Proliferation}, publisher={Wiley}, author={Galand, P. and Degraef, C.}, year={1989}, month=sep, pages={383–392} }

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