Pig coronary artery endothelial cells were cross-linked for 10 minutes at room temperature by adding 37% formaldehyde to the culture medium. The fixed cells were lysed and the chromatin was isolated. For ChIP, sheared chromatin was incubated with Anti-FOXO3A antibody - ChIP Grade (ab12162). The cells were incubated with wortmannin (30 nM), SB 203580 (10 µM) and SP 600125 (30 µM) for 30 minutes before the addition of CGJ for 30 minutes. n = 4 different experiments. *P<0.05 versus control, ^#P<0.05 versus CGJ treatment. Endothelial cells were exposed to CGJ for 30 minutes and then chromatin-bound DNA was immunoprecipitated with an antibody against FoxO3a.

ab12162 staining FOXO3A in Rat fibroblast cells by ICC/IF (Immunocytochemistry/immunofluorescence). Cells were fixed with paraformaldehyde, permeabilized with 0.03% Triton X-100 and blocked with 5% serum for 10 minutes at 25°C. Samples were incubated with primary antibody (1/300) for 1 hour at 25°C.

Immunohistochemical analysis of formalin-fixed, paraffin-embedded Human colon carcinoma tissue, staining FOXO3A with ab12162.

Tissue was fixed with formaldehyde and blocked with 1% serum for 1 hour at 24°C; antigen retrieval was by heat mediation in a citrate buffer. Samples were incubated with primary antibody (1/100 in diluent) for 12 hours at 4°C. A biotinylated goat anti-rabbit polyclonal IgG (1/200) was used as the secondary antibody.

Anti-FOXO3A antibody - ChIP Grade (ab12162) at 1/1500 dilution + Mouse muscle nuclear cell lysate at 40 µg

Secondary
Donkey anti-rabbit HRP conjugate (undiluted)

Developed using the ECL technique.

Predicted band size: 71 kDa
Observed band size: 75 kDa


Exposure time: 1 minute

For protein extraction, murine BaF3^Flt-3,Il-7 cells were washed in ice-cold PBS and immediately resuspended in lysis buffer containing 25 mm Tris (pH 7.5), 1% Triton X-100, 150 mm NaCl, 1 mm DTT, 1 mm EDTA (pH 8.0), 1× protease inhibitor mixture and 1 mm Na3VO4. Samples were centrifuged 1000 × g for 2 minutes and supernatant was mixed with 1× LDS sample buffer and 0.01 m DTT. Samples were heated at 70 °C for 10 minutes before separated by SDS-PAGE and blotted onto a PVDF transfer membrane. Membranes were blocked in 5% milk PBS/Tween or 5% BSA TBS/Tween for 1 hour at room temperature and next incubated with primary antibodies for 1 hour at room temperature or at 8 °C overnight. Following block, membranes were incubated with HRP-conjugated secondary antibody for 1 hour at room temperature. Blots were developed by enhanced chemiluminescence.

ab12162 staining FOXO3A in human brain tissue sections by Immunohistochemistry (PFA perfusion fixed frozen sections). Tissue samples were fixed by perfusion with formaldehyde, permeablized with 0.01% Triton-X 100 and blocked with 5% serum for 4 hours at 25°C. The sample was incubated with primary antibody (1/300) at 4°C for 10 hours. An Alexa Fluor^® 594-conjugated donkey anti-rabbit IgG polyclonal (1/300) was used as the secondary antibody.