Immunohistochemistry and image analysis

At 28 days after MCAO, mice were deeply anesthetized and transcardially perfused with 0.9% NaCl followed by 4% paraformaldehyde in phosphate-buffered saline (PBS). Brains were harvested and cryoprotected in 30% sucrose in PBS, and frozen serial coronal brain sections (25 μm thick) were prepared on a cryostat (CM1900, Leica, Bensheim, Germany). Brain sections were blocked with 5% donkey serum in PBS for 1 h, followed by incubation with primary antibodies for 1 h at room temperature and overnight at 4°C. After a series of washing, sections were incubated with donkey secondary antibodies conjugated to DyLight 488 or Cy3 (1:1000, Jackson ImmunoResearch Laboratories, West Grove, PA, USA). Alternate sections from each experimental condition were incubated in all solutions except the primary antibodies to assess non-specific staining. Sections were mounted and coverslipped with Fluoromount-G containing 4’,6-diamidino-2-phenylindole (DAPI; Southern Biotech, Birmingham, AL, USA). The following primary antibodies were used: rabbit anti-microtubule-associated protein 2 (MAP2; 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), rabbit anti-NeuN (1:500; EMD Millipore, Billerica, MA, USA), rat anti-CD31 (1:200; BD Biosciences, San Jose, CA, USA), rabbit antiglial fibrillary acidic protein (GFAP; 1:500; Dako, Carpentaria, CA, USA). Images were acquired using an inverted Nikon Diaphot-300 fluorescence microscope equipped with a SPOT RT slider camera and Meta Series Software 5.0 (Molecular Devices, Sunnyvale, CA, USA). Alternatively, images were captured with an Olympus Fluoview FV1000 confocal microscope using FV10-ASW 2.0 software (Olympus America, Center Valley, PA, USA).

Chronic brain atrophy was measured on six equally-spaced MAP2-stained sections, encompassing the MCA territory using ImageJ, as previously described [22]. The area of atrophy was measured by subtracting the none-lesioned area (MAP2-positive) of the ipsilateral hemisphere from that of the contralateral hemisphere. The volume of atrophy was calculated by multiplying the mean area of tissue atrophy by the thickness of the tissue evaluated.

The GFAP-stained sections were imaged with fixed exposure setting to quantify the astrocyte scar formation. After randomly selecting three regions of interest (ROIs) from the periinfarct cortex (within 800 μm to the infarct core area), the average intensity of GFAP fluorescence was measured by the NIH ImageJ software. Alternatively, astrocyte scar area was assessed as previously described [23]. Briefly, GFAP immunofluorescence images were converted to black and white and thresholded. The GFAP positive areas were quantified with ImageJ.