Alexa 488 or alexa 568 conjugated goat anti rabbit secondary antibody

To detect intracellular localization of transcription factors, purified Foxp3^GFP+ thymocytes were fixed with 4% PFA at 20 °C for 30 min, permeabilizated with 0.5% Triton X-100 for 5 min at 20 °C, blocked with 3% goat serum (Jackson Immunoresearch) in PBS for 1 h at 20 °C, incubated with anti-Foxo1 (2880P, Cell Signaling) antibody for 1 h, washed with 0.1% Triton X-100 in PBS and then stained with an Alexa 488 (or Alexa 568)-conjugated goat anti-rabbit secondary antibody (Molecular Probes). Alternatively, cells were fixed with eBioscience Transcription Factor Fixation/Permeabilization buffer at 20 °C for 30 min, blocked with 3% goat serum (Jackson Immunoresearch) in eBioscience permeabilization buffer for 1 h at 20 °C, incubated with anti-Foxo1 antibody for 1 h, washed with eBioscience permeabilization buffer and then stained with an Alexa 488 (or Alexa 568)-conjugated goat anti-rabbit secondary antibody (Molecular Probes). Nucleus counterstaining was performed by incubating with 4,6-diamidino-2-phenylindole (DAPI) before mounting slides with Prolong Diamond Antifade Mountant (Thermo Fisher). Images were acquired using a Zeiss LSM 410 confocal microscope.

Immunocytochemistry analysis was performed on rat primary astroglia cultures. Cells were fixed with 4% paraformaldehyde for 10 min, washed with PBS, and permeabilized with 0.1% Triton X-100 for 5 min. Routine staining was performed after a 1-h blocking step in 2% goat serum. Immunohistochemistry analysis was performed on 4 to 6 μm frozen brain tissue sections. After perfusion with 4% paraformaldehyde, dehydration in 30% sucrose, and sectioning, brain slides were blocked with 2% normal goat serum, followed by a routine staining procedure. Monoclonal mouse-anti-GFAP (#556330, BD Biosciences Int. CA, USA) and polyclonal rabbit anti-GBDP (custom-made at Univ. Florida) antibodies at a dilution of 1:200 were used. Alexa 488- or Alexa 568-conjugated goat–anti-rabbit secondary antibody (Molecular Probes, Eugene, OR, USA) was added at a dilution of 1:1000. The cells/tissues were counterstained with 4,6-diamidine-2-phenylindole (Vector Laboratories, Burlingame, CA, USA). Fluorescent images were captured with a 40× objective on the OLYMPUS DP71 fluorescent microscope (Olympus America Inc., Center Valley, PA, USA).