Monoclonal Antibodies to Ki-67 Protein Suitable for Immunohistochemical Analysis

Abstract

Detection of cell proliferation index is widely used in experimental and clinical research. Earlier it was shown that nuclear Ki-67 protein expression is strictly related to cell proliferation. It was revealed during all active phases of the cell cycle in mammals but was absent in G0 phase, so Ki-67 presence in cell nuclei reflects a potential growth fraction of whole cell population. The main area of Ki-67 antibody application is in immunocytochemical and immunohistochemical analyses. The aim of our work was to generate mouse monoclonal antibodies for Ki-67 antigen detection in mammalian tissues and in cultured cells. His-tagged fragment of Ki-67 expressed in bacteria was used as an antigen. Antibody-producing hybridoma cells were generated by standard procedure by fusing SP2/0 myeloma cells with splenocytes of immunized mice. Monoclonal antibodies were analyzed using paraffin-embedded human melanoma tissue samples and breast cancer cell line MCF-7. It was shown that generated anti-Ki-67 antibodies revealed proliferating cells in MCF-7 culture and after heat-induced epitope retrieval on paraffin sections of human melanoma tissue. In summary, generated antibodies could be useful for detection of proliferating cells in immunohistochemical and immunofluorescence studies of mammalian cells and tissues.

mTOR signaling and its involvement in the regulation of cell movements through remodeling the cytoskeleton architecture

“…2). Immunofl uorescent analysis was performed as described [50]. Our fi ndings support a hypothesis that mTOR phosphorilated at Ser 2481 interacts with microtubules during cytokinesis.…”

PI3K/mTOR/S6K signaling pathway – new players and new functional links

“…In addition we found Major components of the PI3K/mTOR/S6K signalling pathway correlation between nuclear accumulation of S6K2 (but not S6K1) and Ki67 and PCNA expression that indicates involvement of S6K2 in malignant cells growth regulation [22]. In support to this observation using in vitro 3D primary monolayer cell culture of thyrocytes, obtained from undamaged follicles, we demonstrated that down regulation of thyrocyte functional activity (dedifferentiation) caused by the loss of follicle organization was accompanied by subcellular redistribution (increase in nuclear content) of S6K1 and S6K2 [24][25][26].…”

Monomethine cyanine probes for visualization of cellular RNA by fluorescence microscopy

“…Afterwords mouse monoclonal antibodies against Ki-67 protein (1:200) were added to the cells and incubated overnight at 4 °C. The anti-Ki-67 antibodies were generated and evaluated earlier [37]. The TRITC-AffiniPure Goat Anti-Mouse IgG (H+L) secondary antibody (Jackson ImmunoResearch Labs, USA) was applied at 1:400 for 40 min at 37 °C in humidified chamber.…”