Alexa fluor 647 anti ly6g

In vivo imaging was performed on the mouse brain 4 h after intracerebral injection of 2 μg of LPS in 5 μl PBS or PBS, as described previously [37 (link)]. Briefly, mice were anesthetized by i.p. injection of a mixture of 200 mg/kg ketamine hydrochloride (Rogar/STB, London, ON, Canada) and 10 mg/kg xylazine (MTC Pharmaceuticals, Cambridge, ON, Canada). The body temperature of mice was kept at 36–37°C throughout the experiment. A craniotomy was performed using a high-speed drill (Fine Science Tools, Foster City, CA, USA), and the durometer was gently removed to expose the underlying pial vasculature. The exposed brain was kept moist with an artificial cerebrospinal fluid throughout the experiment. The mice were inoculated i.v. by the tail vein with Alexa Fluor 647 anti-Ly6G (1A8, BioLegend) to stain neutrophils. Images were taken using a Zeiss Axio Examiner Z1 system. For imaging of the vasculature structure, mice were i.v. injected with 10 μg Alexa Flour 488 anti-mouse PECAM-1 mAb (MEC13.3, BioLegend), as described previously [36 (link)], and euthanized 20 min later. The lung and brain was excised and immediately imaged.