4 6 diamidino 2 phenylindole dapi

Using previously described immunohistochemistry protocols [1 (link),2 (link),3 (link),10 (link)], 20-μm-thick coronal sections were incubated with primary antibodies against nestin (1:200, mouse; Millipore, Billerica, MA, USA) and ionized calcium-binding adapter molecule 1 (Iba-1; 1:500, rabbit; Abcam, Cambridge, UK), followed by incubated with Alexa Fluor 488- or Alexa Fluor 555-conjugated secondary antibodies (1:500; Molecular Probes, Eugene, OR, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:500; Kirkegaard & Perry Laboratories, Gaithersburg, MD, USA). Images were captured using a confocal laser microscope (LSM780; Carl Zeiss, Oberkochen, Germany).
Positive areas for nestin and Iba-1 were evaluated in ischemic and peri-ischemic areas in coronal brain sections obtained from the same region across all animals. A total of 27 data points (3 areas/section and 3 sections/brain, 3 animals/each day) using ImageJ, as previously described [1 (link),6 (link)]. Ischemic and peri-ischemic areas were defined as regions within the borders of stroke-affected areas and regions within a diameter of 100 µm around the stroke-affected areas, respectively, as described [1 (link),6 (link)].

Brain sections (16-μm thickness) obtained from Nestin-GFP mice were subjected to immunohistochemistry using primary antibodies against GFP [1:1000, rabbit, Abcam (ab6556), Cambridge, UK; 1:2000, chicken, Abcam (ab13970)], GFAP (1:500, mouse, Millipore, Billerica, MA, USA), NG2 (1:500, rabbit, Millipore), and PDGFRβ (1:500, goat, R&D systems, Minneapolis, MN, USA). To detect YFP⁺ cells in the brain sections of Nestin-YFP mice, an anti-GFP antibody [1:1000, rabbit, Abcam (ab6556)] that recognizes YFP was used. Bound primary antibodies were visualized using Alexa Fluor 488—or 555-conjugated secondary antibodies (1:500, Molecular Probes, Eugene, OR, USA). Nuclei were counterstained with 4′,6-diamidino-2-phenylindole (DAPI; 1:500, Kirkegaard & Perry Laboratories, Inc., Gaithersburg, MD, USA). Images were captured using a confocal laser microscope (LSM780; Carl Zeiss AG, Oberkochen, Germany) and a microscope (Olympus, Tokyo, Japan).