Immunocytochemical Detection of PCNA and Bax in Cells

Immunocytochemistry was used to detect the presence of PCNA and bax in the cells, as described previously [19 (link),20 (link),57 (link),58 (link),59 (link),60 (link),61 (link)], by means of primary monoclonal antibodies against PCNA and bax (dilution 1:500; from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA), secondary swine antibodies against mouse IgG labeled with horseradish peroxidase (dilution 1:1000; Servac, Prague, Czech Republic) and visualized by DAB-substrate staining (Roche Diagnostics GmbH, Manheim, Germany). In some cases, the assay was validated by these primary antibodies and secondary polyclonal goat antibodies against mouse IgGs, labeled with fluorescein isothiocyanate (FITC, dilution 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA; Figure 1). The negative controls were performed with cells processed without the primary or secondary antibody. The cells were inspected with the aid of a light and a fluorescence microscope (Leica GmbH, Wetzlar, Germany). The cells showing a signal larger than that of the levels of the background negative controls were considered positive. The percentage of cells containing a visible signal–marker of PCNA and bax was calculated relative to the total cell number.

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Sirotkin A.V., Radosová M., Tarko A., Fabova Z., Martín-García I, & Alonso F. (2020). Abatement of the Stimulatory Effect of Copper Nanoparticles Supported on Titania on Ovarian Cell Functions by Some Plants and Phytochemicals. Nanomaterials, 10(9), 1859.

Publication 2020

primary monoclonal antibodies fluorescein isothiocyanate (FITC,

Antibodies Antibody Assay Diagnostics Dilution Fitc Fluorescein Fluorescence microscope Goat Horseradish peroxidase Iggs antibodies Immunocytochemistry Isothiocyanate Light Monoclonal antibodies Mouse Pcna Swine

Corresponding Organization : University of Alicante

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Variable analysis

dependent variables

Presence of PCNA and bax in the cells
Percentage of cells containing a visible signal-marker of PCNA and bax

control variables

Primary monoclonal antibodies against PCNA and bax (dilution 1:500; from Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)
Secondary swine antibodies against mouse IgG labeled with horseradish peroxidase (dilution 1:1000; Servac, Prague, Czech Republic)
Secondary polyclonal goat antibodies against mouse IgGs, labeled with fluorescein isothiocyanate (FITC, dilution 1:1000; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA)
DAB-substrate staining (Roche Diagnostics GmbH, Manheim, Germany)

positive controls

Cells processed with the primary and secondary antibodies

negative controls

Cells processed without the primary or secondary antibody

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