Imagej

This protocol was conducted in a as previously described in Dr. Huang et al. (2017) [24 (link)]. Three cross sections from the middle-third BA in each animal were analyzed by a trained research staff blinded to the experimental groups. The thickness of BA was defined as the largest vertical distance between the inner surface of endothelium and the outer surface of adventitia. The arterial cross-sectional area was calculated using computer-based morphometric analysis (Image J; Universal Imaging Corp., USA). The average area of BA cross sections from each rat was calculated to obtain mean values for the degree of vasospasm at 48 h after SAH.

Mammary gland whole mount analysis was carried out as described by Kleinberg et al. [23] (link). All chemicals came from Merck (Darmstadt, Germany), unless otherwise stated. After harvesting, tissues were fixed in ice-cold 4% paraformaldehyde for 2 h. Mammary glands were stored in 70% ethanol until further analysis. In short, following 3 washes with aceton for 30 min, mammary glands were rehydrated in 100% and 95% ethanol (30 min each). The glands were stained for 1 h with filtered hematoxylin, followed by thorough rinsing with tap water. Background staining was minimized by incubation (3×35 min) in a solution of 150 ml 100% ethanol, 150 ml aqua dest. and 7.5 ml HCl 1N (Sigma-Aldrich Chemie GmbH, Munich, Germany). Subsequently tissues were dehydrated in 70%, 95% and 100% ethanol (2×30 min for each step), followed by an incubation in Xylol (Roth, Karlsruhe, Germany) overnight. For long term storage mammary gland whole mounts were placed in methylsalicylate. Digital images of squash preparations were taken by a transmitted-light microscope stand mounted with a NIKON D200 camera (Nikon GmbH, Düsseldorf, Germany) at a distance of 60 cm all in one day. Picture analysis was performed using MetaVue software (MetaVue, Universal Imaging Corp., Downingtown, PA, USA) and ImageJ [34] (link). Pixel size was 11.125 µmx11.125 µm and pixel area was 123.766 µm².