Alexa fluor 555 conjugated antibody

RAW264.7 cells (3×10⁵/well) were seeded in 24-well plates containing coverslips. Following LPS or BM treatment for 24 h, coverslips were then fixed in 4% paraformaldehyde for 10 min and permeabilized with 0.2% Triton X-100. After blocking in 10% goat serum (DAKO, Glostrup, Denmark) for 1 h at room temperature, the coverslips were incubated with primary NF-κB antibody (1:100; A2574; ABclonal) overnight at 4°C and then with secondary Alexa Fluor 555-conjugated antibody (1:200; 4413s; Cell Signaling Technology) for 2 h at room temperature. Finally, the samples were incubated with 1 μg/ml 4′,6-diamidino-2-phenylindole DAPI (Sigma) for 15 min at room temperature for nuclear staining and detected under a confocal microscope (LSM710; Carl Zeiss, Jena, Germany).

The undifferentiated and the fully differentiated rat AT-MSCs were cultured on two chambers-slide (Nunc^, Rochester, NY, USA). Initially, fixation of the cells was occurred by using 4% paraformaldehyde for 10 min, followed by permeabilization of the cells that occurred with 100% methanol for 10 min. Blocking of the cells by using 5% goat serum was then performed for 1 h at room temperature (RT). Afterward, the cells were overnight incubated at 4 °C with the primary antibodies [mouse monoclonal anti-rat insulin Antibody 1/200 (Novus Biologicals, Littleton, CO, USA) and rabbit polyclonal anti-rat c-peptide antibody 1/100 (Cell Signaling Technology)]. The cells were kept for 2 h at room temperature with the secondary antibodies [1/200 anti-mouse immunoglobulin G (IgG; HL) Alexa Fluor 488 conjugated antibody and 1/100 anti-rabbit (IgG; HL) Alexa Fluor 555 conjugated antibody (Cell Signaling Technology)] after being washed with PBS. The stain [40, 6-diamidino-2-phenylindole (DAPI, Invitrogen, UK)] was used to counterstain the nuclei. No primary antibodies were utilized for the negative controls. The confocal images were taken finally by the Leica TCS SP8 microscope (Leica Microsystems, Mannheim, Germany).