Isolation of Cerebral Blood Vessels from Mice

Cerebral blood vessels of mice were isolated according to Miguel Gama-Sosa’s purification protocol, with some adaptations [35 (link)]. Briefly, mice were sacrificed and their hippocampi were immediately dissected and frozen. Hippocampi of each mouse were gently homogenized in 4 ml of cold 18% w/v Dextran solution in PBS, containing the RNase inhibitors: RNA protect (Sigma, #R7397) and RNA secure (ThermoFisher, #AM7006) and the detergent NP-40 (ThermoFisher, #28324). Homogenization was performed using a Teflon pestle tissue homogenizer and overhead stirrer (10 strokes). Homogenization resulted in a thick homogenate of low density that was overlaid over 5 ml of Ficoll (1.077 g\ml) to form a single-step discontinuous gradient. Centrifugation was performed for 30 min at 400 g and 4 °C. Pellets were then resuspended, washed twice with PBS and the final pellet was suspended in 1 ml Trizol for RNA extraction.