Protocol for isolation of the brain vasculature from the mouse cortex for proteomics

Area of Science:

• Neuroscience
• Cell Biology
• Biochemistry

Background:

• The neurovascular unit (NVU) is crucial for regulating molecular exchange between blood and the central nervous system.
• Understanding the NVU's composition and function requires pure, isolated brain vessel samples.

Purpose of the Study:

• To present a detailed protocol for the isolation of intact brain vessels from the mouse cortex.
• To enable downstream proteomic analysis of the neurovascular unit.

Main Methods:

• Isolation of brain vessels via mechanical homogenization and dextran density-gradient centrifugation.
• Inclusion of steps for cardiac perfusion, tissue preparation, and parenchyma separation.
• Validation of purity using immunofluorescence and western blot.

Main Results:

• Successful isolation of capillary-depleted brain vessels.
• Demonstration of purity through validated methods.
• Preparation of samples for liquid chromatography-tandem mass spectrometry (LC-MS/MS) proteomics.

Conclusions:

• The presented protocol provides a reliable method for obtaining pure brain vessel samples.
• This technique facilitates the proteomic investigation of the neurovascular unit.
• The protocol supports research into blood-brain barrier function and neurological disorders.