HDAC activity assay

To examine the effects of SAHA and MC1568 treatments on HDAC catalytic activity in the ischemic brain, another set of adult male Wistar rats were subjected to MCAO (3 groups, n=6/group). Rats were gavaged with either SAHA (25 mg/kg/d), MC1568 (25 mg/kg/2d) or vehicle (0.5% CMC) for 7 days starting 1 day after stroke. Seven days after MCAO at 2 hours after the last gavage, rats were re-anesthetized with Ketamine (80 mg/kg) and Xylazine (13 mg/kg) via i.p. injection and subjected to cardiac puncture with saline perfusion (approximately 200 mL per rat). Brains were isolated, embedded in cryoprotective OCT solution and flash frozen in 2-methyl butane on dry ice and then stored at −80°C.

A series of cryosections (25 μm thick) were obtained at the center of the lesion, corresponding to coronal coordinates for Bregma −1 to +1 mm (Paxinos and Watson, 2007), and used for protein isolation. Nuclear extracts were prepared using BioVision Nuclear/Cytosol Fractionation Kit (Catalog #K266-25) following the manufacturer’s manual. Protein concentrations were determined by a Pierce™ BCA Protein Assay Kit following the protocol provided by the manufacturer (Life Technologies). HDAC activity was measured on 80 μg of nuclear extract from each sample using a BioVision HDAC activity Colorimetric Assay Kit (Catalog #K331). Samples were read in an ELISA plate reader at 405 nm. HDAC activity is presented as absolute optical density (OD) values obtained from equal amounts of nuclear extracts.