All animal experiments were approved by the Institutional Animal Care and Use Committee of the University of California, San Francisco.
Transient 3 hour MCAO was induced in unsexed P9 mice in a manner similar to previously detailed in P7 rat 22 (link), with modifications 23 (link), are described in the Supplementary Methods.
DWI was performed using a 2T magnet with a Bruker Omega system to identify injured animals and determine the volume of “tissue at risk” ~2.5 hours after MCAO, as described 23 (link).
Genotyping was performed as previously described 6 (link).
Histological methodology, immunofluorescence and 3D data analysis of fluorescence data are described in the Supplementary Methods.
Superoxide production was determined in perfusion-fixated brains following administration of a cell-permeable dye, dihydroethidium (DHE, 5 mg/kg, i.p., 3 hours before sacrifice), as described 17 (link). The total number of Ox-DHE+ cells and the number of Iba1+/Ox-DHE+ and IB4+/Ox-DHE+ cells were determined in 3D-reconstructed images 17 (link).
Western Blot analysis in whole cell lysates, and nuclear and cytoplasmic fractions are described in the Supplementary Methods.
Electrophoretic mobility shift assay (EMSA) was performed to determine nuclear factor (NF)-κB binding activity in nuclear extracts (5 μg/sample) using a commercially available kit (Signosis). The position of NF-κB subunits was confirmed in competition assay by pre-incubating nuclear extracts with non-labeled NF-κB probes (1x-10x dilution). Supershift assay was performed by pre-incubating nuclear extracts with antibodies against p65 and p50 (6 μg/sample; Santa Cruz).
Reverse Transcription-Polymerase Chain Reaction (RT-PCR) is described in the Supplementary Methods.
Chemokine concentrations were measured in injured and contralateral tissue using a LINCOplex™ mouse cytokine multiplex (LINCO Research) as described 24 (link).