Experimental design.

<p>(A) Cranial window in a C57/BL6 mouse. Experimental design of the closed cranial window. (B) Experimental design (upper panel). Representative two-dimensional images of cerebral blood flow measured by laser speckle perfusion imaging before MCAO (a), during MCAO (b), and after reperfusion (c) are shown in the lower panels. MCAO: middle cerebral artery occlusion.</p

Stabilization of POH-N under hypoxic conditions.

<p>SH-SY5Y neuroblastoma cells cultured under normoxic (N) or hypoxic (H) conditions were treated with POH probe. (A) HIF-1α protein levels were analyzed by western blotting (a representative blot is shown). (B) The fluorescence intensity of POH probe in cells was measured. (C) Representative fluorescence images are shown. *<i>P</i><0.02 (vs. normoxic condition).</p

HIF-1α accumulation after focal brain ischemia.

<p>(A) Western blot analysis of HIF-1α in the ischemic and non-ischemic hemispheres of mice subjected to MCAO followed by reperfusion. (B) Densitometric analysis of HIF-1α protein levels in the ischemic hemispheres. Data were normalized relative to β-actin levels, and the values obtained from sham-operated controls (S) were arbitrarily defined as 1. *<i>P</i><0.05 (vs. sham, n = 4).</p

No clear visualization of HIF-1-active regions in the permanent brain ischemia model or with delayed injection of POH-N in the focal brain ischemia model.

<p>(A) Representative <i>in vivo</i> fluorescence images visualized through a cranial window before and at 5 min, 1 h, and 6 h after POH-N administration are shown. POH-N was injected intravenously at 60 min after permanent MCA occlusion. (B) Representative <i>in vivo</i> fluorescence images visualized through a cranial window before and at 5 min, 1 h, and 6 h following POH-N administration at 24 h after reperfusion. Magnified head images are shown in the lower left panels.</p

Imaging of HIF-1-active regions in the focal brain ischemia model.

<p>(A) Representative <i>in vivo</i> fluorescence images visualized through a cranial window before and at 5 min, 1 h, and 6 h after POH-N administration are shown. Magnified head images are shown in the lower left panels. Arrowheads indicate accumulation of the probe in the right ischemic hemisphere. (B) The relative fluorescence intensity of the ischemic hemisphere to the non-ischemic hemisphere. Fluorescence intensities were measured at the indicated times after POH-N administration. *<i>P</i><0.05, n = 3. (C) <i>Ex vivo</i> imaging of the coronal brain sections after POH-N injection. (D) Relative fluorescence of the ischemic hemisphere compared with the non-ischemic hemisphere at 6 h after probe administration (n = 3/group: *<i>P</i><0.05). Relative fluorescence values were calculated using ROIs mirrored along the midline of the cerebral hemispheres. (E) <i>In vivo</i> fluorescence images visualized without preparation of a cranial window before and at 5 min, 1 h, and 6 h after POH-N administration. Anesthetized C57BL/6J mice were shaved and depilated top of the head 24 h before experimentation. Arrowheads indicate accumulation of the probe in the right ischemic hemisphere.</p

Immunohistochemical detection of HIF-1-active cells and POH-N probe.

<p>(A) Immunohistochemical analysis of HIF-1α, POH-N (ODD) and HaloTag (green), with or without DAPI nuclear staining (blue), at 1 day after probe administration. Panels at the bottom show magnified images. (B) Similar distributions of HIF-1α, HaloTag, and HSP70 in pyramidal neurons of the cortical layer bordering the infarct. Scale bars, 50 μm.</p