Microvascular Analysis of Frontal Cortex

We analysed formalin-fixed paraffin-embedded whole coronal sections at levels 6–8 [27 (link), 42 ] containing the frontal cortex (Brodmann area 9). We ensured to select the cortical regions without any obvious infarction. Unless otherwise stated, 2–5 adjacent or alternate whole or half-coronal sections were used for the morphological analyses. Immunohistochemistry was performed to examine different microvascular structures essentially as described before [13 (link), 22 (link)]. The following antibodies were used to assess various cellular features: collagen IV (COL4 at dilation 1:1000, C1926, Merck (Sigma-Aldrich), Branchburg, NJ, USA), a marker of basement membrane in the vessels, platelet-derived growth factor receptor-β (PDGFR-β at 1:200 dilution, clone 42G12, #AF385, R&D systems, Minneapolis, MN, USA), a marker for pericytes, bone morphogenetic protein 4 (BMP4 dilution at 1:100, MBA1049, Millipore, MA, USA), α-smooth muscle actin (αSMA at dilution 1:1000, Clone 1A4, Dako, Cambridge, UK), a marker for mural cells, and glucose transporter-1 (GLUT-1 at 1:200, PA1-21,041, Fisher Scientific, Waltham, MA, USA), a marker of endothelial cells. Vectastain ABC mouse kits (PK-6102, Vector Laboratories, Burlingame, CA, USA) and Diaminobenzidine were used for single or double immunohistochemistry. Haematoxylin counterstain was used for ease in localising regions of interest.