Lsm 700 laser scanning confocal device

Mice were perfused with cold PBS followed by 4% paraformaldehyde 24 h after focal cerebral ischemia. Immediately thereafter, brains were harvested and further fixed for 24 h in 4% paraformaldehyde, following which they were cryoprotected in 30% sucrose for 72 h at 4°C. Each brain was frozen in optical cutting temperature (OCT) compound (Sakura Finetek, Torrance, CA) and stored at −80°C until analysis. The frozen brains were sectioned (thickness: 20 μm) using a CM 3050 cryostat (Leica Microsystems, Wetzlar, Germany). Brain sections were immunostained with anti-ZO-1 (1 : 100), antioccludin (1 : 100, Invitrogen Corporation, Carlsbad, CA), anti-CD-31(1 : 100, BD Bioscience), and anti-AQP4 (1 : 100, Millipore) overnight at 4°C, following which they were incubated with Alexa 488 or Alexa 594-conjugated secondary antibodies (1 : 500, Life Technologies) for 2 h in total darkness. DAPI (molecular probe) was used for nuclei staining. Fluorescence images were captured using a Zeiss LSM 700 laser scanning confocal device (Carl Zeiss, Jena, Germany) and Slide Scanner Axio Scan.Z1 (Carl Zeiss, Jena, Germany). The images are quantified using Metamorph Microscopy Automation and Image Analysis Software (Molecular Devices, USA) and i-Solution software (Image & Microscope Technology, Vancouver, Canada).

All sections (20 μm) were stored in a storage buffer (30% ethylene glycol and 0.01% polyvinylpyrrolidone phosphate-pH 7.4) and were permeabilized for 30 minutes with PBS containing 0.3% Triton X-100. The sections were then incubated in a blocking solution of PBS-T (0.1% tween-20 in PBS) with 10% normal goat serum and were then exposed to the following specific primary antibodies: NeuN (1 : 200, Abcam ab177487, United Kingdom), GFAP (1 : 200, Millipore MAB360, Billerica, MA), Iba-1 (1 : 200, Wako 019–19741, Japan), and cleaved caspase-3 (1 : 200, cell signaling technology 9661L, Beverly, MA). Sections were then incubated with Alexa fluor 488- (1 : 500, Thermo fisher A-11001 and A-11008, Waltham, MA) or Alexa fluor 594-conjugated secondary antibodies (1 : 500, Thermo fisher A-11005 and A-11037) for 2 hours in the dark. We focused on the slices locating at 1,21 and −3.60 mm from the bregma and dashed red square in the cortex indicates the photographed area (Supplementary Figure S1). The fluorescence images were captured with a Zeiss LSM 700 laser scanning confocal device (Carl Zeiss, Jena, Germany), and image analysis was performed using ImageJ software (NIH, Bethesda, MD).