Effect of ionic strength in immunocytochemical detection of the proliferation associated nuclear antigens p120, PCNA, and the protein reacting with Ki‐67 antibody

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Abstract
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 p 120 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. These results also suggest that to enhance the detectability of the intracellular proteins, the current immunocytochemical methods should be modified and customized for each particular antigen in terms of selection of the optimal ionic strength during the fixation procedure.