All animal experiments were conducted in strict adherence to the Swiss federal ordinance on animal protection and welfare as well as according to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC)

All animal experiments were conducted in strict adherence to the Swiss federal ordinance on animal protection and welfare as well as according to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). 1. the complex architecture and physiology of the neurovascular unit (NVU). On the other hand, studies and remains very challenging14. Only a limited number of techniques are amenable to analyze subcellular structures within cells of the NVU. Most studies use electron microscopy but this Loteprednol Etabonate technique is limited by the complex protocols required for proper tissue preparation and sample handling. Therefore, we established a methodology based on high resolution confocal microscopy that would facilitate the processing of brain samples, the analysis, and the quantification of subcellular compartments within cells of the NVU. Here, we describe a protocol which utilizes mouse brain free-floating sections to perform quantitative imaging of the BBB and NVU at the cellular and subcellular levels. We tested and validated a number of antibodies to image and reconstruct the NVU in three dimensions. Furthermore, this protocol allows imaging at the maximal optical IL12RB2 diffraction-limited resolution of organelles within brain capillaries. Together with image analysis, this protocol can be used to investigate the intracellular transport of macromolecules across the BBB under different experimental conditions, for example in mouse disease models of neurodegeneration. Protocol Ethical approval for this study was provided by the Federal Food Safety and Veterinary Office of Switzerland. All animal experiments were conducted in strict adherence to the Swiss federal ordinance on animal protection and welfare as well as according to the rules of the Association for Assessment and Accreditation of Laboratory Animal Care International (AAALAC). 1. Generation of Brain Free-floating Sections To ensure optimal sample quality, prepare a fresh solution of 2% paraformaldehyde (PFA) in phosphate buffer saline (PBS) on the day of the perfusion. CAUTION: PFA is moderately toxic by skin contact and a probable carcinogen. Use nitrile gloves to handle PFA and prepare the solution under a chemical fume hood. Prepare 60 mL of PFA solution per animal. Bring PFA into solution by increasing the pH with 180 – 200 L of a 5 M KOH solution for every 100 mL of PBS and heating the solution up to 60 C. NOTE: NaOH should not be used as it negatively impacts tissue preservation upon fixation. Let the solution cool down to room temperature and bring the pH down to 7.4 using HCl. Filter the solution using filter paper (see Table of Materials). Perform Loteprednol Etabonate a whole mouse fixation via transcardial perfusion as previously described15 with some modifications. First, flush the blood from the vasculature using 20 mL of PBS then perfuse with 40 mL of 2% PFA. Remove the brain from the skull as previously described15. Immerse a freshly 2% PFA-perfused?brain in 20 mL of 2% PFA for 7 h at 4 C for post-fixation. NOTE: Increasing the incubation in PFA is not recommended as it can prevent detection of intracellular structures. Extensively wash the brain with ice-cold PBS. Embed fixed brains in agarose. Prepare a 3% agarose solution in PBS using a microwave for heating. Gently swirl the solution to cool it down but avoid solidification. Dry off the excess of PBS around the brain before immersion into the agarose solution in a plastic container. Rotate the brain inside the agarose to remove air bubbles. Allow the agarose to solidify by cooling it down on ice. Carefully remove the agarose block from the plastic container and cut a cube around the brain using a razor blade.Mount the brain onto a vibratome specimen holder (see Table of Materials) using cyanoacrylate glue (see Table of Materials). Allow sufficient time for the glue to solidify before proceeding with the sectioning. Transfer the brain in the Loteprednol Etabonate specimen holder to the vibratome buffer tray filled with PBS.Use the vibratome to section 100 m brain slices (sagittal or coronal).Collect brain sections in a 6-well plate previously filled with PBS..