NeuN, Iba 1, and GFAP immunohistochemistry

At 1, 2, 3, or 7 days after reperfusion, brains were sliced into 4-mm-thick coronal sections. Sections were then fixed in 4% paraformaldehyde in phosphate buffered saline (PBS) for 2 h at room temperature and cryoprotected with 30% sucrose in 4 °C PBS overnight. Slabs were further sliced in coronal sections (12 μm thick) using a cryostat. For detection of microglia and astrocytes, sections were heated using a microwave oven at 600 W in 10 mM citrate buffer (pH 6) for 4 min to expose the antigen sites. After rinsing in PBS for 5 min, sections were incubated with blocking solution containing 3% bovine serum albumin at room temperature for 1 h, then incubated with mouse anti-NeuN monoclonal antibody (1:500; Merck Millipore, Darmstadt, Germany, Cat No. MAB377) for neurons, or rabbit anti-Iba1 polyclonal antibody (1:500; Wako, Osaka, Japan, Cat No. 019-19741) for microglia, or rabbit anti-GFAP polyclonal antibody (1:500; Dako, Glostrup Denmark, Cat No. Z0334) for astrocytes, at 4 °C overnight. After washing with PBS for 5 min, sections were reacted with Alexa Fluor 488-conjugated anti-mouse IgG antibody (1:500; Invitrogen, Carlsbad, CA, USA, Cat No. A-21202) for neurons, or Alexa Fluor 488-conjugated anti-rabbit IgG antibody (1:500; Invitrogen, Carlsbad, CA, USA, Cat No. A-21206) for microglia and astrocytes at room temperature for 1 h. After washing, sections were mounted using VECTASHIELD Mounting Medium with propidium iodide (Vector, Burlingame, CA, USA) and coverslipped. Immunohistochemical images were obtained using an Axioskop fluorescence microscope. The number of NeuN-positive cells was counted within a box measuring 4 × 10⁴ μm² from the ipsilateral or contralateral dorsolateral striatum and cerebral cortex. The number of Iba 1-positive cells was counted within a box measuring 4 × 10⁴ μm² from the ipsilateral or contralateral dorsolateral striatum and cerebral cortex. For GFAP staining, each section was manually binarized using ImageJ software (National Institutes of Health, Bethesda, Maryland, USA, http://imagej.nih.gov/il/) and the pixel count of a GFAP-positive area within a box measuring 4 × 10⁴ μm² from the ipsilateral or contralateral dorsolateral striatum and cerebral cortex was determined. Data were analyzed under the same conditions between the ipsilateral and contralateral sides and were obtained for one or two sections per animal, from three to four rats.