Anti c fos antibody sc 52

The c-Fos immunohistochemistry was performed as previously described^{53 (link)}. Briefly, anesthetized male mice were intracardially perfused with cold PBS followed by a cold 4% paraformaldehyde (PFA) in PBS. The brains were removed and post-fixed in a 4% PFA solution overnight at 4 °C. Brain regions were cut into 2–4 large blocks. The blocks were sliced on a microtome into 20-μm-thick sections. The sections were pre-incubated in blocking solution (3% bovine serum albumin and 0.3% Triton X-100 in PBS) for 1 h, then incubated with an anti-c-Fos antibody (sc-52, 1:200; Santa Cruz Biotechnology, Santa Cruz, CA, USA) in the blocking solution for 12 h at 4 °C. After three washes with washing buffer, the sections were incubated with goat anti-rabbit IgG antibody coupled with Alexa Fluor 488 (Invitrogen, Carlsbad, CA, USA) in the blocking solution for 1 h at room temperature. The images were obtained by using an Olympus IX71 inverted microscope equipped with a cooled CCD camera (Cool SNAP HQ2; Roper Scientific, Tucson, AZ, USA). The number of c-Fos immuno-positive nuclei in each brain section were recorded and analysed using Metamorph software (Molecular Devices, Downingtown, PA, USA).

Three animals were sacrificed 24 h after ischemia by rapid decapitation under deep anesthesia. Brain tissues were prepared for western blotting. Protein concentration was determined by the Bradford assay (Tiangen Biotech Co., Ltd., Beijing, China). Protein samples (50 μg per lane) were electrophoresed in 10% SDS-polyacrylamide gels and transferred to polyvinylidene fluoride (PVDF) membranes (Millipore, Billerica, MA, USA) at 60 V for 2 hours at 4°C in a transfer buffer containing 48 mmol L^-1 Tris-base, 39 mmol L^-1 glycine, and 20% methanol. The blots were blocked in fresh blocking buffer (Tris-buffered saline with 0.05% Tween 20 [TBS-T] plus 5% non-fat dry milk) for 1 h at room temperature. The blots were then incubated at 4°C overnight with anti-Atf2 antibody (sc-164978), anti-c-Fos antibody (sc-52), or anti-C/EBPγ antibody (sc-25769) (all at 1:1000 dilution; Santa Cruz Biotechnology, Inc., Santa Cruz, CA, USA) and anti-β-actin antibody (1: 10000, Santa Cruz Biotechnology). A secondary antibody conjugated with horseradish peroxidase (HRP, 1:5000, Bio-Rad) was used. Immunoblots were visualized on X-ray film by a chemiluminescence reaction (Pierce, Rockford, IL). Image analysis of the blots was performed on optical density-calibrated images using AlphaEase Stand Alone software (Alpha Innotech Corp., San Leandro, CA).

Nuclear extracts (10 μg) were subjected to SDS–PAGE, transferred to a polyvinylidene fluoride membrane that was blocked with 5% skim milk and then incubated with an anti-c-Fos antibody (sc-52, Santa Cruz Biotechnology, Inc., TX, USA, 1:1000) or an anti-ACTB antibody (#4970, Cell Signaling Technology, MA, USA, 1:1000). Full blotting images corresponding to the immunoblottings shown in the main figures are provided as Supplementary Fig. 1.