Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki-67 antibody.
This study was aimed at revealing whether or not ionic interactions between the epitope of the antigen detected by Ki-67 antibody, or the proliferation-associated proteins PCNA or p120, and neighboring cellular constituents impede detectability of these antigens in HL-60 cells by indirect immunofluorescence assay. To this end, the ionic strength (NaCl concentration) of the solutions in which cells were suspended during their fixation with 0.5% paraformaldehyde was increased, to up to 1.65 M NaCl, to weaken the intra- and/or intermolecular ionic interactions during the process of crosslinking, and the cells were then immunostained. Fluorescence of cells reacting with Ki-67 antibody was maximally increased after their treatment with 1.15 M NaCl; the average increase was nearly 110% above the level seen with the standard methodology utilizing 0.15 M NaCl. The increase was greater for cells in the G1 phase of the cell cycle compared to cells in S or G2. Fluorescence of cells stained with the PCNA antibody was maximally enhanced after cell treatment with 0.65 M NaCl. The enhancement, however, varied depending on the source of the antibody; it was nearly 200% in the case of the antibody provided by Boehringer and over 100% by DAKO. Detection of the nucleolar antigen p120 was not significantly affected by 0.65-1.65 M NaCl. The data indicate that ionic interactions between cellular constituents indeed play a role in masking the epitope of PCNA and the antigen detected by Ki-67.(ABSTRACT TRUNCATED AT 250 WORDS)
Bruno
S
S
Cancer Research Institute, New York Medical College, Valhalla 10595.
Gorczyca
W
W
Darzynkiewicz
Z
Z
D002453:Cell Cycle; D002455:Cell Division; D004305:Dose-Response Relationship, Drug; D005434:Flow Cytometry; D006801:Humans; D007150:Immunohistochemistry; D019394:Ki-67 Antigen; D007942:Leukemia, Experimental / Q000276:immunology / Q000473:pathology; D007951:Leukemia, Myeloid / Q000276:immunology / Q000473:pathology; D009687:Nuclear Proteins / Q000032:analysis*; D009994:Osmolar Concentration; D018809:Proliferating Cell Nuclear Antigen; D012965:Sodium Chloride / Q000494:pharmacology; D014407:Tumor Cells, Cultured / Q000276:immunology / Q000473:pathology; D012359:tRNA Methyltransferases
D019394:Ki-67 Antigen; D009687:Nuclear Proteins; D018809:Proliferating Cell Nuclear Antigen; D012965:Sodium Chloride; C065028:NOP2 protein, human; D012359:tRNA Methyltransferases