Rabbit monoclonal anti c fos

The reagents used were bought from the following specified manufacturers: rabbit monoclonal anti-c-fos, cleaved caspase 3, caspase 3, rabbit polyclonal anti-Pim-2, horseradish peroxidase (HRP)-anti-rabbit IgG, and anti-mouse IgG from Cell Signaling Technology (Beverly, MA, USA); mouse monoclonal anti-β-actin antibody from Sigma-Aldrich (St. Louis, MO, USA); mouse monoclonal antibodies against NFATc1 and CTSK from Santa Cruz Biotechnology Inc. (Santa Cruz, CA, USA); human macrophage colony-stimulating factor (M-CSF) from Wako (Osaka, Japan); human soluble RANKL from Oriental Yeast Co. Ltd (Shiga, Japan); febuxostat from TEIJIN (Osaka, Japan); NAC from Nacalai tesqure (Kyoto, Japan); and Dox from Tokyo Chemical Industry (Tokyo, Japan).

Dissociated cells grown on coverslips were fixed in 2% PFA in PBS for 5 minutes, then transferred to 4% PFA for an additional 15 minutes and then washed with PBS. Unspecific binding of antibodies binding was blocked with 7% normal donkey serum and 0.3% Triton diluted in PBS for two hours at RT. Overnight staining was performed at 4°C with primary antibody diluted in 2% bovine serum albumin with 0.05% azide and 0.1% Triton. Thereafter, cells were washed three times with PBS and incubated with 1 μg/ml DAPI (Sigma-Aldrich), Alexa Fluor647- and DyLight488-coupled secondary antibody (Biomol, Hamburg, Germany) diluted in 2% bovine serum albumin with 0.05% azide in PBS for two hours at RT. Coverslips were washed in PBS and specimens were mounted with Fluoromount (Sigma-Aldrich). The following antibodies were used: Rabbit monoclonal anti c-Fos (Cell Signaling Technology, Inc., SanDiego, CA, USA) 1:800, Rabbit monoclonal anti γH2A.X (phosphor S139) antibody (Abcam, Cambridge, UK) 1:400, guinea pig polyclonal anti c-Fos (Synaptic Systems, Goettingen, Germany) 1:500. Imaging was done on an Olympus IX81 epifluorescence microscope (Olympus Life Sciences, Germany) or on a spinning disc confocal (Microscope (VisiScope 5-Elements, Visitron Systems, Germany) and images were subsequently analyzed with ImageJ or Matlab.