Fig. 2. Immunolabelling to visualise developing blood vessels in the mouse embryo hindbrain.

An E12.5 hindbrain was labelled by PECAM immunohistochemistry, flatmounted and imaged at the indicated magnifications. (a-c) Flatmounting the hindbrain with the pial side up allows visualisation of radial vessels entering the brain. (d-f) Flatmounting the hindbrain with the ventricular side up allows visualisation of the subventricular vascular plexus. The dotted boxes in (a,d) indicate the areas shown at higher magnification in (b,e), the dotted boxes in (b,e) those shown at higher magnification in (c,f), respectively; the size of each field in (c,f) is 500 μm × 500 μm, i.e. 0.25 mm². (g,h) Counting of radial vessels and vascular intersections in the fields shown in (c,f); green dots were used to track vessels that have been counted. (i-k) A 100 μm transverse vibratome section through the E12.5 hindbrain shown in (a); radial vessels (rv) extend from the pial side of the hindbrain and then branch to form the SVP below the ventricular side of the hindbrain; (j,k) higher magnification images from both sides of the hindbrain shown in panel (i). Scale bars: (a,d) 1 mm; (b,e,i-k) 200 μm, (c,f) 100 μm. All animal procedures were performed in accordance with institutional and UK Home Office guidelines.