Computational protocol: Cellular selectivity of AAV serotypes for gene delivery in neurons and astrocytes by neonatal intracerebroventricular injection

Similar protocols

Protocol publication

[…] Images of transduced primary cells and all quantitated AAV infected brain regions were acquired using a 20X air objective with a Zeiss Axiovert 200M inverted fluorescent microscope equipped and a Hammatsu ORCA-ER-cooled charge coupled device camera (Hammatsu Pho-tonics, Hammatsu City, Japan). For IF quantitation, multiple random, z-stack images of approximately 30 μm dissector height for both hemispheres of all brain regions (in vivo quantitation) or a single 2D images (for in vitro quantitation) were acquired per coverslip/brain region and counted total %GFP cell/image field using Slidebook software (version 5.5, Intelligent Imaging Innovations, Denver CO). A background subtraction was performed for all IF in vivo images, prior to quantitation. Representative high-magnification images were acquired with a Zeiss Plan-Apochromat 100X oil objective lens. Representative 10X objective montage images of ROI were acquired with a BX51 microscope (Olympus, Center Valley, PA, USA) equipped with a Hammatsu ORCA-Flash4.0 digital CMOS camera, ProScan III stage controller (Prior, Rockland, MA USA) and CellSens Dimension software (version 1.12, Olympus, Center Valley, PA, USA). All bright field images of mock-injected brains were acquired with Olympus SZX12 stereo-dissecting microscope. For enhanced CLARITY fluorescence imaging of AAVDJ8-GFP, 40X oil objective montage images were acquired using a Fluoview 1200 scanning-laser confocal microscope to penetrate ~80μm thickness of tissue (Olympus, Center Valley, PA USA). CLARITY fluorescent imaging of AAVDJ8-GFAP-mCherry was conducted on a Zeiss LSM 510 Laser-scanning confocal microscope to penetrate and capture ~180μm thickness of tissue. Both sets of CLARITY images were 3D-rendered in ImageJ analysis software []. Representative images of whole brain fluorescence were acquired with ChemiDoc MP imaging system (BioRad). Uninjected controls were imaged with each serotype for comparison of background fluorescence (Fig C in ). […]

Pipeline specifications

Software tools cellSens, ImageJ
Applications Laser scanning microscopy, Microscopic phenotype analysis
Organisms Adeno-associated virus, Adeno-associated virus - 2, Mus musculus
Diseases Brain Diseases, Infection, Parkinson Disease, Neurodegenerative Diseases