Formamide

Cytosolic dsDNA and DNA-RNA staining were performed as described previously (16). Briefly, 50% formamide (VWR International, USA) diluted in PBS was added for 10 mins at room temperature, followed by incubation for 15 mins at 75°C to denature dsDNA. Cells were washed with PBS and incubated with blocking buffer (1% BSA (Sigma Aldrich), 2% goat serum (Hyclone) in PBS) for 1 h to prevent non-specific binding of antibodies. Cells were then stained with dsDNA (1:200, Sigma-Aldrich, USA) or S9.6 DNA-RNA hybrids (1:100, Kerafast, Boston, USA) antibodies overnight in 4°C. Cells were subsequently washed with PBST (0.1% Tween) thrice followed by anti-mouse IgG coupled with Cy3 (Millipore, Singapore) or anti-rabbit IgG coupled to Alexa Fluor 488 (Invitrogen, Singapore). Finally, DNA fluorophore DAPI (0.5 μg/ml in PBS, KPL Inc., USA) was added for 10 min. Slides were washed once in PBS before mounted with Da-/- fluorescent mounting medium (Da-/-, UK). Confocal images of staining were captured using a Zeiss Axio Imager Z1 fluorescent microscope equipped with AxioVision 4.8 software (Carl Zeiss MicroImaging, USA) or confocal TCS SP5 (Leica, Singapore). Images were analyzed using Photoshop CS4 (Adobe, USA) or ImageJ. Colocalisation of AIM2 with DNA or other proteins were quantified using Metamorph (Metamorph NX, version 8.12, Molecular Devices, USA).

The chemical reagents used in the study were as follows: deoxyadenosine 5′-triphosphate, [α-³³P] (NEG312H; NEN Research Products, PerkinElmer, Waltham, MS, USA); CaCl₂, NaCl, KCl (Merck, Whitehouse Station, NJ, USA); aminoguanidine hydrochloride, Denhardt's solution, dithiothreitol, N-lauroylsarcosine, polyethylene glycol 300, β-mercaptoethanol, phenylmethanesulfonyl fluoride, S-adenosyl-methionine (Sigma, St Louis, MO, USA); MgCl₂ and (Riedel-deHaёn, Seelze, Germany); formamide (Amresco, Solon, OH, USA); RNA (Roche, Basel, Switzerland); and dextran sulphate (Amersham, Amersham, UK).

The BBB permeability was assessed by examining the extent of Evans blue (EB, Sigma, USA) solution leakage from the microvessels in the rat brains following intravenous injection. Briefly, Evans Blue dye (2% in saline, 4 mL/kg) was intravenously administered via tail vein and allowed to circulate for 60 min. To clear the blood and intravascular Evans blue solution that remained in the vascular system, all the rats (n = 10 per group at each experimental time point) were deeply anesthetized with 10% chloral hydrate and perfused with heparinized saline through the cardiac ventricle until colorless perfusion fluid was obtained from the atrium at 5 h, 24 h, 72 h after pMCAO. After decapitation, the hemispheres of brain were separated along the interhemispheric plane. For quantitative measurement of EB, the cortex of the ischemic hemispheres were weighed and incubated in formamide (1 ml/100 mg, Amresco) at 54 °C for 24 h and samples were centrifuged at 3000 rpm for 15 min. The supernatant was collected, and the OD at 620 nm was measured using a 722-type spectrophotometer (Shanghai Third Analytical Instrument Factory, Shanghai, China). The amount of Evans Blue in the tissue was quantified using the standard curve. Data was expressed as μg/g of brain tissue.