Sc 5258

For immunofluorescence staining, MSCs were grown in cover slips, washed with PBS, fixed with methanol, permeabilized with 0.1% Triton X- and blocked with 5% BSA in TBST. The samples were incubated with the following antibodies overnight at 4 °C: glucocorticoid receptor (D6H2L, 1:200, Cell Signaling), HDAC6, Runx2 (SC-5258 and SC-390715, 1:50 Santa Cruz). The appropriate secondary antibodies used are: Alexa Fluor 488-conjugated goat anti-rabbit, Alexa Fluor 586-conjugated goat anti-mouse, Alexa-fluor 688- conjugated donkey anti-goat (Life technologies) at a 1:250 dilution in PBS and counterstained with DAPI (1:2,000; Invitrogen Corporation, NY, USA). Images were acquired from at least 50 cells per treatment using Olympus Ax80 microscope. For co-localization detection, images were obtained using a confocal microscope (Zeiss LSM 700) and processed using LSM Image Browser (Zeiss).
For western blot, cells were harvested and lysed using RIPA buffer, and immunoblots were probed with glucocorticoid receptor (D6H2L, Cell Signaling) and Runx2 (SC-390715, Santa Cruz) antibodies.

Brains were homogenized in RIPA buffer (50 mM Tris·HCl, pH 7.5, 500 mM NaCl, 2.5 mM MgCl₂, 1% NP-40, 10% glycerol) containing proteinase inhibitors; 30 μg of protein was separated by SDS/PAGE and transferred to PVDF membrane for immunoblot analyses. The membranes were blocked with 5% nonfat dry milk in PBS containing 0.1% Tween 20 (PBST) and probed with primary antibodies recognizing acetylated lysine (1:2000; rabbit monoclonal; Abcam (ab190479), Cambridge, UK), α-tubulin (1:1000; rabbit polyclonal; Cell Signaling Technology (2144S), Danvers, MA), and HDAC6 (1:100; goat polyclonal; Santa Cruz Biotechnology (sc-5258), Dallas, TX). Membranes were incubated with primary antibody overnight at 4 °C, washed three times with PBST, followed by incubation with horseradish peroxidase-conjugated anti-rabbit (Jackson ImmunoResearch (111–035-144), West Grove, PA), anti-goat (Jackson ImmunoResearch (111–035-144)), or anti-β-actin antibodies (Sigma-Aldrich (A3854)) at room temperature for 2 h. Membranes were then washed three times with PBST. Immunoreactivity for each protein was visualized using a chemiluminescence detection kit (GE Healthcare Life Sciences, Little Chalfont, UK). The images were acquired using ImageQuant LAS 4000 (GE Healthcare Life Sciences) and densitometrically analyzed using ImageJ software.