Anti mouse igg hrp sc 2005

Raw264.7 cell lines were cultured in DMEM (Gibco) supplemented with 10% FBS, 100 U/ml penicillin, 100 μg/ml streptomycin and 10 μg/ml gentamycin. One day prior to treatment, 1 x 10⁶ cells/well were seeded on a 6-well plate. Cells were treated with 0, 0.2, 0.4, 0.8 mg/ml of lidocaine for 2 h and subsequently stimulated with LPS for 10 min. Cells were washed with cold PBS, lysed with NP–40 lysis buffer containing protease inhibitor cocktail (GenDepot) and incubated with continuous agitation at 4°C for 30 min. After centrifugation at 13,000 g for 15 min, supernatants were taken and 30 μg of protein were used for SDS-PAGE. Protein samples were transferred to Immobilon-P PVDF membrane (Millipore). The following antibodies were used for western blot analysis: anti-IκB-α (L35A5, 1:1000 dilution, Cell Signaling Technology), anti-β-actin (AC–15, 1:5000 dilution, Abcam), anti-mouse IgG-HRP (sc–2005, 1:5000 dilution, Santa Cruz Biotechnology).

A frequently used marker of trophoblastic cell fusion is the FI^{46 (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 counted^{19 (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.