Computational protocol: Epidermal growth factor prevents APOE4-induced cognitive and cerebrovascular deficits in female mice

Similar protocols

Protocol publication

[…] General protocol. Frozen brains were sectioned at 12 μm and fixed in 10% neutral buffered formalin (Sigma). Slides were incubated in 52.8% formic acid (8 min), permeabilized with TBS containing 0.25% TBSX (3 × 5 min), blocked with 5% BSA (2 h), incubated with primary antibodies (4 °C, overnight), washed (3 × 5 min in TBSX), incubated with secondary antibodies (2 h), washed with TBSX (3 × 5 min) followed by TBS (1 × 5 min), and mounted. Nine nonadjacent sections (108 μm apart) were used for quantification per animal.Fibrinogen. Fibrinogen (Rabbit anti-fibrinogen 1:200 from Dako, AlexaFlour 647 anti-rabbit 1:200 from Invitrogen) was co-stained with CD31 (Rat anti-CD31 from B&D Bioscience with AlexaFluor 405 anti-rat for characterization studies of 8 month old mice, and AlexaFluor 488 anti-rat for the EGF study, both at 1:200 from Invitrogen). 6 images from each cortex per section were captured on a Zeiss Axio Imager M1 under identical capture settings at 20x magnification. For representative images, Z-stack images were taken at 25x magnification on a Zeiss LSM 710Confocal Microscope and 3D reconstructions were produced using Imaris 7.7.2 software.Laminin. Hemi-brain sections were imaged for laminin (rabbit anti-laminin 1:400 from Abcam, AlexaFlour 750 anti-rabbit 1:200 from Invitrogen) using the Zeiss Axio Mosaic setting. Quantification was performed on the deep layer isocortex and full isocortex.Quantification. Images were thresholded equally to diminish background signal (NIH ImageJ software) and quantified using the Analyze Particles function. […]

Pipeline specifications

Software tools Imaris, ImageJ
Application Microscopic phenotype analysis
Organisms Mus musculus, Homo sapiens
Diseases Alzheimer Disease, Hypertension, Liver Diseases