Sc 1004

Paraffin sections (5 µm) of paraformaldehyde-fixed eyes of albino Balb/c mice and frozen sections (14 µm) of paraformaldehyde-fixed eyes of C57BL/6J mice were used for immunohistochemistry.²⁵ (link) We used rabbit polyclonal antibody against Adrb2 (1:2000, bs-0947, Bioss), glucocorticoid receptor (GR) (1:200, sc-1004, Santa Cruz Biotechnology, Dallas, TX), guinea pig polyclonal antibody against Per1 (1:200, PM091, MBL, Nagoya, Japan) and mouse monoclonal antibody against Bmal1 (1:1000, sc-365645, Santa Cruz Biotechnology, Dallas, TX), at 4°C for 48 hours. This was followed by incubation with biotinylated goat antirabbit IgG (120 minutes; 1:1000; PK-6101, Vector Laboratories, Burlingame, CA), and then avidin–biotin complex solution (120 minutes; 1:400; Vectastain Elite ABC Kit, Vector Laboratories) before substrate visualization using 3,3′-diaminobenzidine (349-00903, Dojindo, Kumamoto, Japan). For immunofluorescence, sections were incubated with donkey polyclonal AlexaFluor 488-conjugated antirabbit IgG (1:400; R37118, Thermo Fisher Scientific, Waltham, MA) and goat polyclonal DyLight 549-conjugated antiguinea pig IgG (1:400; 606-142-129, Rockland Immunochemicals Inc, Pottstown, PA) secondary antibody for 120 minutes (1:400; R37118, Thermo Fisher Scientific).

BMMФ were incubated -/+100 nM Dex for 40 min and ChIPs were performed as in [23 (link)] using N499 [22 (link)] and sc1004 (Santa Cruz Biotechnology) anti-GR antibodies, or normal rabbit IgG as a background control. The data for each recruitment site was normalized to non-specific signals at the unrelated 28S ribosomal gene. Primer pairs are listed in (Additional file 2: Table S3). ChIP-seq is detailed in (Additional file 1).

PBMCs were incubated with dexamethasone (1 µM) or 1, 25(OH) 2 D3 (100 nM) for 30 minutes. PBMCs were immunostained for glucocorticoid receptor antibodies (1:500 dilution) (sc-1004, Santa Cruz Biotechnology). Tetramethylrhodamine isothiocyanate (TRITC)–conjugated anti-rabbit secondary Abs or fluorescein isothiocyanate (FITC)-were used to probe the primary Abs. The nucleus was stained with DAPI reagent. The slides were analyzed with a Leica TCS SP5 confocal microscope (Leica, Germany).

In short, ChIP was performed as described previously [66 (link)] with minor modifications. Frozen livers (approximately 50 mg per IP) were homogenized in 1% Formaldehyde PBS solution using Ultra-Turrax homogenizer on level 5 for 10 seconds, with following cross-linking at room temperature for 10 minutes. The suspension was quenched by adding 0.125 M glycine and incubating additional 10 minutes at room temperature. Cross-linked cells were washed in PBS, resuspended in lysis buffer (0.1% SDS, 1% Triton X-100, 0.15 M NaCl, 1 mM EDTA, 20 mM TrisHCl [pH 8.0], and BSA 1 mg/ml), and sonicated using Bioruptor (Diagenode) or ME220 Focused-ultrasonicator (Covaris). Chromatin was immunoprecipitated over night at 4 °C using antibodies and Protein A/G agarose beads (Santa Cruz, sc-2003). H3K27Ac ChIP was done with 0.2 μl/IP of H3K27Ac antibody Ab4729 (Abcam). GR ChIP antibody cocktail consisted of 1 μg/IP of MA1-510 (Thermo Fisher), 1 μg/IP of PA1-511a (Thermo Fisher), and 1μg/IP of sc-1004 (Santa Cruz). FOXO1 ChIP was done with 3 μg/IP of sc-11350 (Santa Cruz). Immunocomplexes were washed, and chromatin was eluted (1% SDS with 0.1 M NaHCO₃) and decross-linked over night at 65 °C. DNA was phenol/chloroform purified and ethanol precipitated. Recovery was analyzed by qPCR using primers listed in S2 Table and/or sequenced.