Limitations in the measurement of c-myc oncoprotein and other nuclear antigens by flow cytometry.
Recent developments in cell fixation, quantitative immunocytochemistry, and flow cytometry allow for the quantification of a variety of oncoproteins and other proliferation-associated antigens in both fresh and archival pathology material. These studies provide evidence that the standard tissue deparaffinization/dissociation technique significantly reduces the amount of c-myc oncoprotein remaining for analysis. To examine the factor(s) responsible for this observation, individual variables of the deparaffinization/dissociation technique including type of fixative, pepsin concentration, pepsinization times, pH, and exposure to organic solvents were examined in HeLa-S3 cells. The cells were stained with monoclonal antibodies either to the c-myc oncoprotein or to p105, a prolifera-tion-associated nuclear antigen. Protein-levels were measured on the basis of anti-c-myc or anti-p105 immunofluorescence by flow cytometry and were found not be affected significantly by type of fixative, exposure to organic solvents, acid pH solution, or mechanical disruption. Levels of c-myc oncoprotein were reduced by over 50%, however, when cells were exposed to 0.5% pepsin, whereas p105 was more resilient with only an approximately 7% reduction following the same treatment. Thus, careful examination of aspects of the deparaffinization/dissociation technique appears to be a necessary prerequisite for quantification of specific nuclear proteins from dissociated tissue specimens.
Lincoln
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Department of Pathology, Northwestern University Medical School, Chicago, Illinois 60611.
Bauer
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D000911:Antibodies, Monoclonal; D005404:Fixatives; D005434:Flow Cytometry / Q000379:methods*; D006801:Humans; D009687:Nuclear Proteins / Q000032:analysis*; D011518:Proto-Oncogene Proteins / Q000032:analysis*; D016271:Proto-Oncogene Proteins c-myc; D014407:Tumor Cells, Cultured / Q000032:analysis*
D000911:Antibodies, Monoclonal; D005404:Fixatives; D009687:Nuclear Proteins; D011518:Proto-Oncogene Proteins; D016271:Proto-Oncogene Proteins c-myc