A frequently used marker of trophoblastic cell fusion is the FI46 (link). It describes the number of nuclei inside syncytia as a percentage of the total number of nuclei.
Trophoblastic cell FI was determined by immunocytochemistry, as previously described by Bastida-Ruiz et al.16 (link). Briefly, cells were washed in PBS, fixed in 3% paraformaldehyde and immunostained using mouse anti-desmoplakin antibodies (sc-390975, 1:500 dilution from Santa Cruz Biotechnology, Heidelberg, Germany). Revelation was performed with diaminobenzidine (Dako, Carpinteria, CA, USA) after incubation with secondary antibodies anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotechnology, Heidelberg, Germany). Nuclei were stained with haematoxylin. Image acquisition was completed using a Ceti Inverso TC-100 inverted biological microscope (Medline scientific, UK). FI was expressed in percentages and calculated as follows: [(N − S)/T], where N equals the number of nuclei in syncytia, S equals the number of syncytia, and T equals the total number of nuclei counted19 (link). The syncytia are recognised as cells containing several nuclei inside the same cell. Three different fields were analysed per well for each experiment. FI was calculated for at least three independent experiments, run in triplicate.
Trophoblastic cell FI was determined by immunocytochemistry, as previously described by Bastida-Ruiz et al.16 (link). Briefly, cells were washed in PBS, fixed in 3% paraformaldehyde and immunostained using mouse anti-desmoplakin antibodies (sc-390975, 1:500 dilution from Santa Cruz Biotechnology, Heidelberg, Germany). Revelation was performed with diaminobenzidine (Dako, Carpinteria, CA, USA) after incubation with secondary antibodies anti-mouse IgG-HRP (sc-2005, Santa Cruz Biotechnology, Heidelberg, Germany). Nuclei were stained with haematoxylin. Image acquisition was completed using a Ceti Inverso TC-100 inverted biological microscope (Medline scientific, UK). FI was expressed in percentages and calculated as follows: [(N − S)/T], where N equals the number of nuclei in syncytia, S equals the number of syncytia, and T equals the total number of nuclei counted19 (link). The syncytia are recognised as cells containing several nuclei inside the same cell. Three different fields were analysed per well for each experiment. FI was calculated for at least three independent experiments, run in triplicate.
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