Donkey anti rabbit apc

To harvest MG d0, the brains of P1–3 inbred Lewis rats (Orientbio, Sungnam, Korea) were minced and the CD11b⁺ cells isolated using MACS without a mixed glial cell culture. The MG d14 were isolated from the mixed glial cell culture and trypsinized. Both groups of cells were blocked with 1% bovine serum albumin (BSA, Sigma, St. Louis, MO, USA) and incubated with primary antibody for 30 min at 4 °C: CD11b (BD Bioscience, San Jose, CA, USA, 1:100), CD206 (Abcam, Cambridge, MA, USA, 1:200), CD163 (AbD Serotec, Oxford, UK, 1:100), and CD68 (Millipore, Burlington, MA, USA, 1:200). After washing the cells with 0.1% BSA solution, the cells were incubated with secondary antibody for 30 min at 4 °C: APC donkey anti-rabbit (Jackson ImmunoResearch, W Baltimore Pike, PA, USA, 1:500) and FITC goat anti-mouse (Abcam, Cambridge, MA, USA, 1:500). The data were acquired with the BD FACSDiva 7.0 software.

After treatment with dexamethasone (10⁻⁷ M) for 1 h, the cells were fixed with 4% formaldehyde for 20 min, permeabilized, and blocked. The cells were then incubated with the primary antibodies (mouse monoclonal antibody to GR; Abcam, Cambridge, UK and rabbit polyclonal antibody to PP2A; GeneTex, Alton Pkwy Irvine, CA, USA), followed by the fluorescently labeled secondary antibodies (Alexa 488 donkey anti-mouse and APC donkey anti-rabbit; Jackson Immuno Research, West Grove, PA, USA). Control antibodies and Hoechst (Invitrogen, Paisley, UK) were included in each experiment. GR’s ability to translocate into the nucleus was evaluated using a Carl Zeiss LSM700 confocal microscope.